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Questions related to Molecular Diagnostics
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We have a Qiagen QIAquant PCR instrument at COVID 19 Molecular Diagnostic Lab and we have used standard 8-well strips 0.2 ml so far. Now we are going to utilize standard 96-well microplate. Is there anybody worked with these microplates? Can we reuse the plates? If yes, after finishing a run, How should we clean up the plate and prepare for the next run? and How many times can we use them? Finally, I should mention our work is so sensitive because we do diagnostic tests.
Thank you so much
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Katie A Burnette Thank you for your kindly response. It was so useful. This was our problem. I didn't think that we can cut the plates. Thank you again for sharing us this important matter. Good luck and have a nice day.
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Does Autoclaving and gamma irradiation remove DNA enough so that no DNA fragment will be amplified in PCR?
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you can try treating your glassware with DMSO treated water
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Molecular methods are widespread in modern zoology. And I'm not sure if they are universal enough to be applied in all scientific fields.
Are examples of failure of molecular diagnostic systems for identification in your work?
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Molecular diagnostic systems has been identified as sensitive as well as specific for species identification by many researchers. But due to high cost, standardization, contamination of samples etc failure rate is also very common. .
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Dear colleagues, we (with my collaborators) develop an interesting multi-wallet carbon based nanosensors for for amplification-free DNA detection. According to literature this kind of sensors is non very popular but there are enought evidence of their potential in molecular diagnostics. May be anybody use (used) nanosensor in their work? Could you give me feedback? We would like to improve and adopt our sensors for clinical diagnostics.
Now we able to recognize SNP's and specific viral/bactrerial NA in samples after routine column-based NA purification. We do not use any amplification. The detection is non-fluorescent.
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Dear Andrei
In best of my knowledge it's really better for your team to start comparative study of your sensor with others are using in Food safe grade to show benefits and advantages of your sensor.
I think it's easiest and the best step to start.
Then step by step take more challenging issues because even in clinical sensors are not welcome yet.
According to math, physics and chemistry sensors are sensors like photons detectors but you can see easily even we don't believe nonodrop for example.
So you have a long way to go.
With best wishes.
Alireza
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I am interested to explore the Genetics of Complex Human Diseases (infectious diseases and cancer). Aiming to evaluate the transcriptional and proteomics regulation of genes/ proteins involved in disease progression and proliferation, Molecular insight of Host-pathogens interaction, genetic variation and drug-resistance mechanisms, molecular diagnostic, prognostic and predictive Biomarker of the diseases.
Being an early career researcher I have submitted several projects for funding based on molecular biology but no successful results yet. I am still trying very hard to get funds and collaborate with national and international researcher.
I have an expertise at OMICs level (DNA/RNA/Proteins techniques), if you have any related interest, you are welcomed for collaboration, we can share the present resources or can wright a joint research projects, submit somewhere for funding....
Hope to work together for the betterment of our coming generation...
Best Regards
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Dear @Najeeb Ullah Khan
Thanks for the wishes.
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Studying the sensitivity of methods used in HLA typing
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It's very important question
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We are on the path of developing Genotype panels for QC evaluation of molecular diagnostic kits (HIV, HBV, HCV). What would be a better method to approach - Sanger sequencing method or NGS platform. The sample size is tentatively 1000 numbers.
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Abhijeet Singh, If you work in a lab that is funded by grants, cost is always a concern. Sanger sequencing has higher initial investment on sample processing and preparation, but the sequence analysis is fairly simple, just basic alignment. NGS however, you can just prepare the RNA and send out. However, you do need capability to analyze the high-throughput data afterwards. For that, you can not completely rely on others to do the analyses for you, not just for concern of the cost, in my opinion. Otherwise, it will be very inconvenient when it comes to write the manuscripts, every little change you want to make, you'll have to contact the people that did the initial analyses and rerun them.
Chandranand: Theoretically, TI-NGS is definitely applicable for your experiment design. However, you do need consider the sequencing depth if you need the full length sequence of the target genes. If you just need the matched counts of the pathogen genes, that will be very straight forward.
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I have developed RT PCR mastermix , now its required to add stabilizer in it? if yes which stabilizer we can use at which concentration.
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Hi Maulik,
Good to have your own master mix rather than commercial. In general, glycerol, BSA, Triton-X 100, Betaine, DMSO, formamide etc are often used in conventional and real time PCR as an additive. I would like to know your buffer system whether it is KCL or Tris HCL. Based on the buffer system the concentration of additives or stabilizers will differ. However, above mentioned additives are widely used. Better, run the experiment with your basic buffer system and ingredients. Then, based on the result the choice of additive and concentration can be selected. As a general condition most of the buffer system contain BSA (1-10 mg/ml) and Glycerol (2-5%). Most of the amplifications occurs well in these concentration.
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From the methods I have come across, I haven't seen any that claim to be capable of purifying nucleic acid from bacterial, fungal and viral cells. Is there a commercially available method out there capable of this? Also, most methods seem to focus on blood. Are there any methods out there for sputum? Thanks for your help.
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Check this paper! It can give you some ideas.
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Molecular diagnostic of BPH Nilaparvata lugens (Stal) biotypes.
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By knowing the different biotypes of BPH present, we will be able to use present resistant varieties accordingly
And can reduce selection pressure on BPH.
If I am wrong plz correct me sir.
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A couple seeking premarital genetic counseling:
Male: heterozygous for ∆3.7 single gene deletion mutation (alpha thalassemia trait)
Female: heterozygous for IVS II-1 G>A mutation (beta thalassemia trait)
What would be the phenotype of a kid carrying both traits?
Thank you in advance.
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Double heterozygote or alpha+ and beta thalassemia will have less globin chain imbalance than trait of either of them; and so, the clinical and hematological picture will be better than both the parents. Read an interesting case at: Blood Cells, Molecules, and Diseases 32 (2004) 319 – 324
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molecular diagnosis
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NGS sequencing of a clinical exome gene panel (TS1) and analysis limited to 8 genes associated with PKD (BICC1, LRP5, NOTCH2, PKD1, PKD2, PKHD1, PRKCSH, SEC63). If the potential candidate variant is detected in PKD1 gene (it has 6 highly homologous pseudogenes) then it is confirmed by long-range PCR of selected region, followed by exon-specific Sanger sequencing. If the variant is detected in any other gene it is confirmed by classic PCR followed by Sanger seq.
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My laboratory is trying to develop a qPCR assay to detect rodent fur mites Radfordia affinis and Radfordia ensifera. However, there are no published nucleotide sequences in NCBI for either species, nor publications with primer/probe sequences in Pubmed.
Thank you for your help in advance.
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Hola, José
Did you find information about PCR primers for Radfordia affinis? Take a look at this:
Maybe it can help you.
I'm also looking for a way to detect radfordia affinis... that is the only thing that I've found.
If you have something else, please let me know.
Regards
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We are about to start our diagnostic lab for Fragile X, and the real-time machine that appeals to us is LineGene 9600 Plus (https://www.biosan.lv/en/products/molecular-diagnostics/amplificationpcr/linegene-9600-plus-real-time-pcr-detection-system)
Its specifications are satisfactory. However, we could not find any feedback.
So if any of you has worked with this model, please, let us know if it is worth purchasing it. What are pros and cons that you have encountered while working with this qPCR machine. 
Your feedback may turn out to be decisive for us.
Lela
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Hello good day!
The descriptions are very close to LineGene, but have more filters.
I liked the optical system, efficient and highly reliable, including for diagnostic kit validation.
If you go with a large scale diagnosis, I suggest the Q7 flex.
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I had already quantified my DNA using a Spectrophotometer, and most of the samples were around the 200-300 ng/ul. When I quantify using Picogreen assay, my standards range from 10-1000 ng/ml. I know the conversion is 1000ng/ml= 1ng/ul... So, if one of my samples concentration with the picogreen is 600ng/ml that means I only have .6ng/ul?? 
I have been trying to find out about this, maybe I'm not doing the conversion right? Please, someone help. 
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If you go for Spectrophotometer reading even for water adding some junk it gives reading.
Just load by taking 1 microliter of your DNA on the gel then do visual quantification, by comparing the readings that you got by Spec. I say that is better. Then proceed for other things like dilutions etc. etc. 
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i want to know the genetic disease in india for which molecular diagnostics is used the most
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Please. What is the molecular mechanism of statins in Pathological Erythrocytosis of Height?
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I want to avoid chaotropic reagents as they are supposedly toxic and as I am developing a point of care diagnostic protocol for ZIKA to be used on the field (not under normal laboratory conditions), I would prefer a non-toxic reagent.  
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Are you looking to isolate ZIKA RNA from serum??  Perhaps try using Kosmotropic salts which work by increasing water surface tension rather than disrupting water structure to encourage hydrophobic interaction hence protein precipitation.
Here's a paper that decribes isolation of RNA from E.coli using this method.  How suitable this might be for Viral RNA isolation I've no idea......good luck
Analytical Biochemistry
Volume 381, Issue 1, 1 October 2008, Pages 160-162
Isolation of total RNA from Escherichia coli using kosmotropic Hofmeister salts
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Can anyone tell me what type of chemical reactions happens in diagnostic kits like- pregnancy detection kits? From where I can get details about similar kits used in diagnostics?
I want to have list of such ready to use reliable diagnostic kits. Whomever knows any just reply in answer. Thanks.
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In diagnostic kits like- pregnancy and HIV detection kits; there is a kind of immunological reaction; in which the kit might coated with invisible either antigen or an antibody.  During serological detection of pregnancy for example, the kit is usually coated with ant-hCG antibody and we wre going to look for the hCG hormone from urine of pregnat women. The working principle of most kits is just like this.
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I have to be prepared if the virus comes up in Europe or Germany.
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Thanks a lot Stefan, I'm already in contact with Avi Eldar.
best wishes
Sven
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Hi
I would like to know why beta globin is used in real time PCR for HPV detection?
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Beta globin or other house keeping genes, such as RNase H, encode proteins that are involved in the basic function of cells, therefore they are used as indication of the quality of swabs collected. That is, if enough cells (quantitative detection of the gene, hence the use of qPCR) were collected. Since HPV is ubiquitous, it is essential to show that a sample of collected swab that is positive for HPV also had enough cells from which the HPV detected had infected. Therefore, a qPCR of a house keeping gene is essential to confirm that the HPV detected was from the sample.
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During specificity test of a diagnostic, when it challenged against plasmid DNA from a non-target species at a very high concentrations of DNA non-target (10E8 copies/ul) there was amplification. However, at lower concentrations this did not occur. Non-specific amplification may have occurred due to stochastic events, ie, since all the components necessary for amplification to occur occur (except for the target DNA), the physical interaction between the molecules could have favored non-specific amplification, considering this extreme situation of High concentration of non-target DNA?
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In short, yes, it can happen.  
In real-time PCR assays that are designed to be very sensitive you can sometimes have amplification of a nonspecific target.  There are several possibilities.  Real-time PCR assays, although often more specific than conventional PCR assays because of additional specificity provided by the probe.  However, some assays will tolerate one or more mismatches in the probe and/or primer sequence.  One possibity, then is amplification of a phylogenetically related organism that has a similar sequence.  
To find out whether you have nonspecific amplification of a single template, I would recommend sequencing the PCR product as a first step.  Also, if you find that you are amplifying a nonspecific target, you can redesign the assay to exclude that nonspecific target and you can sometimes slightly improve specificity by adjusting the cycling parameters, such as by slightly increasing the annealing temperature to improve specificity, depending on the annealing temperature of your primers and probe.
Best regards,
Marcus
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Idea about universal primer of low risk Human Papilloma Virus? Only  universal low risk Primers?
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good luck
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Can Cas9/gRNA recognize SNP? What is the sensitivity of Cas9/gRNA? If the sensitivity is not good, is there any enzyme in Cas family can recognize SNP? or have higher sensitivity? Thanks!
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However, if this single nucleotide mutation or the SNP leads to the forming of  a novel protospacer adjacent motif (PAM), then CRISPR/Cas9 should be able to distinguish this PAM-containing allele from other alleles.
See the attached paper: CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting 
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The delivering time of the dig-labeling kit(Roche) is so long. Is there any other effective ways to label the RNA fragments with digoxin ?
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I need to detect and quantify simultaneously 6 different bacterial gene from stool sample. Is it possible by using Applied Biosystems™ TaqMan® QSY ™ probe to optimise the required qPCR in BioRad CFX96 system.
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I have multiplexed successfully with Fast Advanced Mastermix and Environmental Mastermix. I believe the CFX96 will only multiplex 5 targets though... The QSY probe is non-fluoresent so can be used on different probes in a  multiplex reaction. I have used QSY to replace BHQ quenchers in several assays.
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Is there any research/groups working into miniaturizing AC Pulsed Field Gel Electrophoresis? Like if you want to use it a lab-on-a-chip or similar formats.
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Alternative to PFGE, diversiLab system is the product of bioMérieux. You can find details in the link. 
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How effective and reliable would "Ion AmpliSeq™ Cancer Hotspot Panel v2" be for identifying variants of oncogenes and tumor suppressors genes in xenograft tumor? 
Expecting comments from users of this panel. 
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sure, the interest for such panels is to have a deep view on the samples (I mean with high coverage, for cellular clones for example). if you look the MET exon14 coverage, you'll see that only variants at 5' will be covered, forgetting 50% of the published variants in the 3' of this exon...this panel is a good base but you can adapt it to what you observed in your lab.
fred
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I made an in-vitro transcription of a nuceoprotein gene (cloned in pGEM-T) to determine the viral load in infected patients with RT-qPCR assay. After obtaining the apropriate quality and quantity of RNA, the calibration curve was obtained with serial dilutions of the transcript.
When I made the calibration curve, those dilutions more than -8 (with a Ct=30) resulted negative in the RT-qPCR. However, there are a few samples of infected patients that are detected by this RT-qPCR with Ct values more than 30 (eg. 33, 36). Therefore, I cannot determine the viral loads of those samples considering that my calibration curve doesn't include Ct values more than 30.
Do you have any idea why this happens and how can I quantify these problematic samples?
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Hi Matías,
Purified standards and biological samples (with matrix effect etc.) seldom amplify at the same efficiency for the same target of interest. As well, some plasmid constructs (if not linearized prior to deploying into the RT-qPCR) can stall the onset of amplification for 5 or more cycles. Assuming some of this may be at play here, the following could apply:
I assume you are saying that your standard dilution of 10-8 gives you a Cq value of 30. And I assume you may be doing serial 1:10 dilutions for your standard curve, thus, in order for the next Cq value in the standard series to not appear (no signal/negative RT-qPCR), this would imply that 1 or less copies of target standard in the reactions is being indicated after that dilution.  This could be a feature of poorly efficient reactions. But, for now, let's assume that 1 copy should amplify at ~38.32 Cq, and that your standard curve efficiency is 100%.
On the other hand, the samples from your patients could be amplifying right away (without initially stalling the RT-qPCR). And let us also assume that patient sample amplification is also ~100% E or EAMP = 2 and has an expected 1 copy intercept value of about 38.32 Cq. Since the patient samples may be more amenable to immediate amplification in RT-qPCR, this could be giving you more sensitivity with the patient samples (e.g., beyond a Cq of 30 in your present case). Why your plasmid standard curve signal falls off into the abyss after a Cq of 30 is not yet readily explainable without more information.
Taking this all one step further, if your plasmid standard curve Cq value of 30 really indicates about 320 copies of target strand in a RT-qPCReaction (at 100% reaction efficiency and a y-intercept 1-copy estimated Cq of 38.32), then patient samples amplifying at 100% efficiency (also with an estimated intercept Cq of 38.32) at 33 and 36 Cq would indicate about 40 and 5 copies in those patient sample RT-qPCReactions, respectively.
Next thing to consider is the virus:  whether or not it goes through a DNA phase or not, or, e.g., if it is just a negative or positive sense single-stranded RNA virus etc.  You will want to clarify what 'one target copy' is to reach the appropriate parity with what your standard curve indicates.  Your standard curve plasmid construct is likely ssRNA after thermal breakage (which is theoretically complete after the 5 cycle stall; if not linearized beforehand), but your patient samples may include both DNA and viral RNA NP as targets in the RT-qPCR - both of which would be amplified after the RT phase is complete (if your virus also goes through a DNA stage in vivo that is).
Your original question is leaving out key details - most of which I've had to guess at here.  But, in order for me to answer better, what are your amplification efficiencies of 1.) The plasmid standard curve, 2.) a standard curve made from positive patient samples, and 3.) how many copies do you claim are represented at each of your standard dilutions, 4.) How do you quantify copies of your RNA plasmid transcript standard up front, and do you linearize the plasmid transcript prior to RT-qPCR, 5.) Are you making certain the RNA standards are not degraded before deployment into the RT-qPCR. 6.) Provide Cq data from your standard curve (e.g., log of copies vs Cq).
PS Cq = "quantification cycle"  (formerly called "Ct"; threshold cycle).
The next problem to explore will be the eventuality that you will find that the plasmid and the patient samples do not amplify target at the same efficiency.  These two universes can be reconciled in theory by an algorithm I came up with in 2011.
Without more information, for now, these are good things to consider.  Bear in mind, it is largely impossible to ever get 1 target copy in a qPCR reaction to amplify earlier than ~35 cycles (usually it is more like 37 or 38 cycles at 100% efficiency). That being said, it appears to be inferred with the results you've discussed in your original question, that you may be dealing with a very poorly efficient standard curve.  E.g., if you have signal at 30 Cq, but no signal at  (an assumed) 1:10 dilution later, this means that a Cq of at least 35 (at 1:10 dilution later) means you are dealing with a standard curve amplification efficiency of about 58.48% or lower...
~
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Anyone know what is the minimum concentration (copies/uL) that should obtained?
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1.32 ng/µL that you have in your PCR you should be getting around 380 copies/µL in the ddPCR
If your target is homozygous and the 20ng/µL is accurate, then you should get around 1000 copies/µL.
Note from JacobMclaughlin
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Hey,
In order to standardize and optimize positive controls for Real-time PCR in our laboratory, and also to gain time, we were wondering the stability of plasmid dilutions for semi to long term use in our diagnosis routine.
We want to know if we can store our positive controls dilutions at 4°C for a month without loosing any stability ?
I'll appreciate any knowledge or experience or feedback on that subject
cheers.
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Thanks for the kind answers,
I asked for a little bit more precision about purification of plasmids from the company that does this job for our laboratory.
They told me the plasmids were purified with the kit NucleoSpin Plasmid, Macherey Nagel, Ref. 740588250 in accords with the instructions of the kit.
I guess that means that they are minipreps and should be story at -20°C for long time storage (1-2months) ? Any feedback or experience would be really appreciated. 
thank you in advance for your answers.
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I am planning to perform Methylight to bisulfite-converted cell-free DNA isolated from blood(Serum/plasma). Can you please suggest which DNA Polymerase with 5' to 3' exonuclease activity works best with cell-free DNA? (I will use Stratagene Mx3005p Real-Time PCR system)
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Thank you for your suggestions.
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Can anyone give me the exact THP protocol for DNA extraction. I have been already using this based on the following reference, but unable to optimize it yet.
Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal
DNA from Maternal Serum Zeinab Keshavarz, Leili Moezzi.
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you can follow ATCC protocol 
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So the DNA is extracted then converted via bisulphite treatment before the PCR.
Wondering is there a good method for optimisation of the PCR before i move on to clinical samples.
Thinking spiking known pos cells in a negative background or spiking converted DNA into a non converted DNA background
Thanks
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Dear Stephen,
It is better to optimise your PCR method before moving on to the clinical samples.  Hence I would recommend you to perform PCR analysis initially using positive control samples followed by hybridization technique or even a nested PCR would give better results.
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What are the methods to reduce PCR bias. which is the technique having the least PCR bias?
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I didnt get you.
Can you please specify the things out?
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HCV genotyping using conventional methods
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Well, You can take a look of this article, might be you can get an idea based on instrument availability:
"HCV Genotyping from NGS Short Reads and Its Application in Genotype Detection from HCV Mixed Infected Plasma"
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Hi 
If I have 200-1000 SNPs I need to test on regular basis with clinically validated way ( direct-to customer service) which way I may choose e.g. custom made Microarray or Taqman probes ( I need the cost as least as possible not exceed 100$ for 200 SNPs if possible).
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I published the book about microarray. See RG HP.
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-DNA from Urine
- Toxoplasma Gondii
- LAMP
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 The Amount of DNA ranged from 19 to 7182 ng/ml for the female urine and from 6 to 7128 ng/ml for the male urine.
But its depends on many physiological condition.
What is the concentration of DNA in urine? - ResearchGate. Available from: https://www.researchgate.net/post/What_is_the_concentration_of_DNA_in_urine [accessed Jan 29, 2017].
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currently PVL is detect by PCR only, and its fatal toxin which need early detection specially among inpatients and those in ICUs
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International Journal of Food Microbiology 138 (2010) 287–291
this paper can help you.
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Does someone knows a good book or a PDF file on the subject of DNA repair? Especially with BRCA1 as a key gene? It is for a thesis defence. So it shouldn't be too extensive. 
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never mind - and thank you for your help...!!!
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In search of an adequate prognostic model for early recognition of delrium in Cardiothorasic ICU patients
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Certainly electrolytes (eg hypo/hypernatraemia, hypokalaemia) and liver enzymes (eg cirrhosis/hepatic encephalopathy) can cause acute brain syndromes, let alone hypoglycaemia, sepsis etc (see Gen Hosp Psychiatry. 2014 Mar-Apr;36(2):156-64). However we have found that routine Vitamin B12 testing was not very useful in predicting post-operative delerium.
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I performed Mycobacterium marinum genomic DNA (mmar_2193) using specific primer (FP-ttcatatggattttgatgcgctg and RP 1-ttaagcttctagttttgccgccgcgc and RP 2-ttaagcttgttttgccgccgcgc) 
PCR master mix
5x Reaction buffer- 5ul
GC enhancer        - 5ul
dNTP                     - 1ul
DNA                       - 1ul
FP                          -1ul
RP                         - 1ul
miliQ water              -31.5
q5 polymerase       -.5 ul
total                        - 50 ul
PCR Cycle
980c   980c  640c*  720c     720c      40c
 2min   30s  45s    30s    10min        ~
1cycle           30cycle           1 cycle
i use gradient angling temperature 63-65 °C.
but all time comes nonspecific band. My band size 636bp.
what did i do wrong?
please suggest to me! 
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Nonspecific PCR reaction  may be related to inappropriate cycling, very short primers, Incorrect PCR mix . Try to check step by step with positive and negative control
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I did COLD-PCR for JAK2 wildtype and mutant samples. The previous results were okay, the peaks were around 76-77 celcius. Now the peaks are around 77-78, I wonder what happened. My samples are kept in -80 celcius freezer, I have tapped and spinned down them and my master mix before running them on RotorGene.
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As the other respondents have indicated, this is likely to be an effect of minor changes in buffer conditions, possibly due to inaccurate pipetting. The possible reasons are many. A leaky seal in one of your pipettes is one possibility. 
Note, however, that the PCR reaction itself will change the buffer conditions, due to the release of inorganic phosphate from the dNTPs. This causes the Tm to drift slightly as the concentration of amplicon (and thus Pi) increases. 
As long as the Tm's for your samples match those of your controls, this should not be a big problem, but keep an eye on it, as it might be a sign of other problems on the horizon. 
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Hi, I ran two gels couple of days ago and did not get good bands. I initially thought that it was my secondary that's not working well. But when I checked the gels I realized that there were thick bands at the low molecular weight marker levels.
Every time I do transfer I would check if the ladder is transferred onto the blot (that's how I determine if the transfer is successful). The ladder was on the blot but why are the proteins not transferring??
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Hi Elena,
As Fernanda lead off with, small proteins usually transfer fastest.  It is possible you are allowing the transfer to proceed for too long and your proteins are going out the back side of your membrane.
You can try decreasing time of transfer or sticking additional membranes behind your intended membrane to see if they are going too far.
Good luck!
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Is there a particular feature or a direct connection that can explain why immunohistochemical expression of mutant protein p53 is increased in obese and diabetics (type 2 diabetic) patients at diagnosis of colorectal adenocarcinoma compared to non -obese diabetics versus non-diabetics obese/non obese?
Study included immunohistochemical analysis of  2 proteins Bcl2 and p53 (both monoexpression and coexpression) in 100 newly  diagnosed colorectal adenocarcinoma specimens divided  equal in 2 groups diabetics (type 2 diabetes mellitus already existing at diagnosis of colorectal cancer)  and non-diabetics .The preliminary statistics results have not indicated a significant difference between the 2 groups, in terms of Bcl2, but  indicates an increased expression of p53 only in the  diabetic-obese group compared to  non-diabetics obese or non-obese (statistically significant ) which theoretically indicates a contact point between obesity, cancer and diabetes. Interestingly moderately low degree of differentiation is associated with both diabetes and the presence of p53 positivity; G2-p53 associations is an intriguing part. Bcl2 expression was low and insignificant in both group.
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Proper regulation of white and brown adipogenic differentiation is important for maintaining an organism's metabolic profile in a homeostatic state. The recent observations showing that the p53 tumor suppressor plays a role in metabolism raise the question of whether it is involved in the regulation of white and brown adipocyte differentiation. By using several in vitro models, representing various stages of white adipocyte differentiation, we found that p53 exerts a suppressive effect on white adipocyte differentiation in both mouse and human cells. Moreover, our in vivo analysis indicated that p53 is implicated in protection against diet-induced obesity. In striking contrast, our data shows that p53 exerts a positive regulatory effect on brown adipocyte differentiation. Abrogation of p53 function in skeletal muscle committed cells reduced their capacity to differentiate into brown adipocytes and histological analysis of brown adipose tissue revealed an impaired morphology in both embryonic and adult p53-null mice. Thus, depending on the specific adipogenic differentiation program, p53 may exert a positive or a negative effect. This cell type dependent regulation reflects an additional modality of p53 in maintaining a homeostatic state, not only in the cell, but also in the organism at large.
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How to estimate the hpv antigen content by ELISA test ?
What type of elisa kits are using for estimation of hpv antigen content and equipment .
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Hi Nataraja,
It depends on the gene of interest and the type of HPV, knowing that the L1 ORF is the most conserved among all papilomaviruses.
I suggest the PCR assay
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I am having problems getting any detected insulin from dried blood spots after extraction with PBS+Tween.
I am using a common biochemical analyzer (electroluminescence) instead of ELISA (which could be the problem since ELISA detection has been successful according to bibliography).
Any ideas?
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I really dont know of a way to extract without hemolysis
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I am working on  Molecular diagnostics of Human Papilloma Virus through Conventional PCR, but there is no Internal Control on the Kit which I am using for it so I just want to use human beta globin gene primer with the master mix of that kit but i don't have any idea regarding this. Can anyone help me out of this......... 
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Extracted from the reference sheet of one I use in my lab, hope it helps:
This product is a set of PCR primers for amplification of β-globin region
of human genome DNA. By combining two of these primers, nine differ-
ent lengths of DNA fragment can be amplified. Therefore, these primers
can be used as a positive control in PCR reaction when confirming
whether template DNA is appropriate as PCR template.
Components : each 500 pmol
1.PC03 (forward) d (ACACA  ACTGT  GTTCA  CTAGC) 
2.PC04 (reverse) d (CAACT  TCATC  CACGT  TCACC) 
3.GH20 (forward) d (GAAGA  GCCAA  GGACA  GGTAC) 
4.GH21 (reverse) d (GGAAA  ATAGA  CCAAT  AGGCA  G)
5.KM29 (forward) d (GGTTG  GCCAA  TCTAC  TCCCA  GG)
6.KM38 (reverse) d (TGGTC  TCCTT  AAACC  TGTCT  TG)
Reconstitution:
Reconstitute by adding sterilized distilled water or TE Buffer (pH7.5 - 8.0).
Reconstituted solution should be kept at -20℃ .
β-globin (human) Primer set
Size:500 pmol x 6 Shipping at RT
Store at - 20℃
Amplified fragment (bp) :
reverse
PC04 GH21 KM38
forward
PC03 110 250 167
GH20 268 408 325
KM29 205 345 262
PCR condition :
94℃ 1 min
55℃ 2 min 25 - 35 cycles
72℃ 1 min
Reference :
Saiki R.K., Gelfand D.H., Stoffel S., Scharf S.J., Higuchi R., Horn G.T., Mullis
K.B. and Erlich H.A. (1988) Science, 239, 487-491.
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We have diagnosed a girl with McKusick-Kaufman syndrome (MKS) based on clinical presentation with hydrometrocolpos, polydactyly, and congenital heart disease. We want to confirm the diagnosis at the molecular level, but we do not know where to do this.
Has anyone suggestions about laboratories to perform mutational analysis of MKKS gene? 
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Usually we calculate the force constants for bond length, bond angle and dihedral angle. I want to calculate the force constant for entire molecule.
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It makes no sense to speak of the force constant for an entire molecule as a single scalar quantity. Rather it will be a matrix of quantities depending on the molecular geometry and number of bonds. See the attached lecture series  on modelling systems of springs.In simple linear series and parallel spring systems you can calculate overall constants but not generally.
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I want to quantify Fe, Cu, Ni, Sn and Cd from urine. What are the procedures for posterior analysis of the sample in ICPMS? Should I acidify the urine?
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The follow article is perfect for you research, Cheers.
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2
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In most cases LAMP is monitored with non-specific indicators such as turbidity, SYBR green, hydroxynaphthol blue etc.  As such it can be prone to detecting non-specific amplification.  In practice this depends on the primer set - some primer sets are more prone to "false positives" than others.  Amplification time also makes a difference - the longer you let it go, the more likely to get a "false positive".  I also find (at least for RT-LAMP) that it can be a little less sensitive than corresponding qRT-PCR.
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i want to start molecular diagnosis unit in my lab. so i need highly specific kits for different genetic disease. I don't have Real-Time PCR, I just have conventional gradient PCR machine, so i need that kits only which can work with it.  For Qualitative analysis ( Diagnosis) only.
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In a basic setting, reverse dot blot technique can be useful: machines are quite inexpensive and allows to make optimal diagnosis without an rtpcr machine or a sequencer. There are excellent kits and you need just a pcr machine (autoblot machine is optional, can be done free hand).
Hope it helps.
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- sample 9 has a negative result using exactly the same mastermix I used for the negative control. 
- used a 100 bp molecular weight ladder
- negative control: water
- 9-14 are the samples 
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As long as you have a band in the negative control you have to fight contamination. Use fresh solutions, use a different set of pipettes, ... Lanes 10 to 14 could be positive, but there is no proof. Sample 9 could be negative due to some inhibiting activity in your samples not present in the water control.
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Hi
I am doing cDNA subtractive hybridization to know the differential expressed genes using Clonetech Kit.  First, I did PCR using random primer, but did not get any amplification and then I did PCR utlizing nested primer for 3 different hybridized sample.  The 1st and 2nd samples are diluted where as the 3rd is not diluted.  I have used Emerald Master Mix for PCR and loaded 5 microliter.  I have attached the gel picture, please give your valuable suggestion.  Thanks
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Subtractive hybridization is a bit out-dated and very labor intensive.  A more modern way to look at differential gene expression is using microarrays such as Affymetrix or Illumina, or next generation sequencing of whole transcriptomes, i.e. RNA-Seq.  This requires some specialized equipment but these methods can also be done as a fee for service through a variety of companies.  Just send them your RNA.   The great advantage is that these methods cover most of the genome and are quantitative as well. 
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miRNA is proposed to be clinical biomarker in many diseases, as well acute and chronic ones. I think that there is a need to validate extraction protocols to introduce miRNA into molecular diagnostics as a valuable parameter .
There are many commercial kits and in house methods you can recommend to extract miRNA from body fluids, but it there is always a problem to obtain good concentrations of miRNA to perform high-throughput methods or even e sequencing. There are some protocols published but I want to activate people who REALLY deal with clinical samples, and can share their authentic experience.
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Hello to all,
This is my first time working with miRNAs and I am trying to isolate them from plasma samples. I don't know why but the service that is going to measure the miRNAs is concerning about A260/280 ratios and I have not read anything about it here. I am working with miRCURY Biofluids from Exiqon and I am obtaining 15 ng/microl with 500 microl of plasma but my ratios are about 1.6-1.7. Is this normal? Thanks!!
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We tried several times to isolate RNA from HK-2 cells with the qiagen RNeasy kit (with RLT buffer). We can do the isolation of RNA (with Qiagen kit) from other lines of human immortalized proximal tubular epithelial cells without any problem, but from the HK-2 cells it's very hard to get RNA. We already tried to homogenize the sample better, using needle and syringe. We also already added the RLT buffer immediately to the culture dish (instead of to the pellet) and scraped the cells. All without improvement...
What else could we try?
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Use the  trizol method as Dr. Murthy indicates. There are several commercial denominations but all are the same product essentially. I used it some time ago with HK-2 cells and didn't have any problems with it.
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Please suggest steps for detecting amplification by loop mediated isothermal amplification (LAMP) assay using lateral flow dipstick (LFD). The FIP is biotin labelled and probe is FITC labelled since i am using LFD from Milenia.
I was able to get very faint test line by incubation of probe (Tm 67) with amplicon at 65 degree celcius for 5 minutes. But the result is non-reproducible.
I have tried various concentration of probe and different hybridization temperature based on published literature. I have tried with fresh reagents also. There is no signal on test line despite very high amplification.
Please suggest how to troubleshoot the problem or how to carry out LAMP assay with LFD.
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Have you tested if the problem is in the isothermal amplification reaction or in the LFD? I mean, after isothermal amplification (I gess 65ºC during 30-60 min) have you tested (by rt-pcr or agarosa gel) if the amplification reaction is working properly?
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What will be the consequences out of this?
Extract by Florent Mouliere / Montpellier and Nitzan Rosenfeld / Cambridge:
Circulating tumor DNA (ctDNA) is now widely investigated as a biomarker in translational and clinical research. However, despite the growing field of clinical applications, the biology of ctDNA remains unclear. In trying to learn about the origins of ctDNA, nature provides us with very few clues. One of the important accessible parameters is the size of those DNA fragments. In addition, a well-informed model of these sizes and biases can help design more efficient and accurate diagnostic methods. In PNAS, Jiang et al. take an important step in that direction.
Significance
We used massively parallel sequencing to study the size profiles of plasma DNA samples at single-base resolution and in a genome-wide manner. We used chromosome arm-level z-score analysis (CAZA) to identify tumor-derived plasma DNA for studying their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These findings have shed light on fundamental biological characteristics of plasma DNA and related diagnostic applications for cancer.
Abstract
The analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for the assessment of solid tumors. Although its clinical potential has been increasingly recognized, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. With respect to the size profile of such plasma DNA molecules, a number of studies reported the finding of increased integrity of tumor-derived plasma DNA, whereas others found evidence to suggest that plasma DNA molecules released by tumors might be shorter. Here, we performed a detailed analysis of the size profiles of plasma DNA in 90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B, 36 with hepatitis B-associated cirrhosis, and 32 healthy controls. We used massively parallel sequencing to achieve plasma DNA size measurement at single-base resolution and in a genome-wide manner. Tumor-derived plasma DNA molecules were further identified with the use of chromosome arm-level z-score analysis (CAZA), which facilitated the studying of their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of plasma mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These results have improved our understanding of the size profile of tumor-derived circulating cell-free DNA and might further enhance our ability to use plasma DNA as a molecular diagnostic tool.
Jiang P et al.: Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients. Proc Natl Acad Sci U S A. 2015 Feb 2. pii: 201500076.
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well pointed out.
We also have this discussion at the web group of theTheodor-Billroth-Academy on LInkedin and Reinhold Kiehl, Hossein Rahmati Roudsari and Jan Voskuil made very important points and I try summarizing those:
-tumor markers as CEA or CA19-9 should be used for follow-up-never for making the diagnosis, as the overall accuracy is too small. Further tumor markers are expressed proteins on surface membranes and we learned from epithelial cysts that such can mimick a cancer, e.g.in spleen cysts very high CA-19-9 had been observed without any association of any cancer, nor during follow-up
Therefore, we by now do not know 
- the influence and value and accuracy of detectable DNA
-when the best time is measuring it and how the values are influenced
-how storing would be best and how the interobserver variability might be
-when free DNA is measurable, how do such values differ after treatment
-are they proven being related to the observed cancer or assumed to be
-microRNA#s had been observed in ling cancer patients assuming having impact on spread and even cancer development but again: assumed!
-to my knowledge no standard protocol is accepted or suggested determining free DNA so far
Additionally Ijaz Jamall added a valuable insights deepening he comment from Hossein Rahmati Roudsari and Jan Voskuil (copy and pasted):
Ijaz Jamall: "I agree with Dr. Rousdari and Jan: one key factor to consider in measuring anything in the blood is its half-life and how to "normalize" the thing being measured - per ml blood or serum, per g protein, etc. because essentially we are measuring a concentration as opposed to a mass and dilution and binding kinetics or changes in hydration or albumin content can make huge a difference. In fact, diurnal variation can itself make a huge difference"
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I am new to the field of circulating nucleic acid detection. I would like to search for microRNA down/upregulation in blood samples from patients suffering from certain type of cancer. I have never tried to isolate and quantify extracellular micro-RNA. Have you tried it? Maybe there is a good kit you can recommend? Is the 30-5-5 min serum centrifugation necessary?
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Hi Filip,
you need as little as 300 µl of plasma. If you want more amount of RNA, you need only to increase proportionally the amount of buffers in the kit.
Here you have the workflow (page 2): 
2) If you want to try another method that works well, you can lyse the plasma with TRIzol LS and then purify RNA with this kit from ZYMO research (Direct-Zol): 
3) For obtaining plasma, we centrifuge blood samples for 15' at 1000g, and then, place the plasma aliquoted in 1'5 mL tubes and  freeze immediately at –80ºC.
Ginés
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One of my collegues accidently put the silica columns out of the refrigerator about six months ago. I have known that the binding efficacy will be dropped in this situation but, is there any one who experienced similar situation like this?
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Use some antibiotic on top of the column bed and seal it from lower and upper side and keep it at room temperature. There will be no problem up to 27oC temperature.
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I have dissected out mouse hippocampus and want to homogenize it for analysis of phosphorylated and aggregated tau. We have dounce homogenizers but they feel too big for such small tissues. We also have a TissueLyser from Qiagen (normally used for RNA-prep) which uses a bead to disrupt the tissue, but I have been told that too strong mechanical disruption can destroy the large aggregates. Are there any do's and don't-s for this type of sample preparation? 
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as suggested by Monica glass or platic pestle homogenizers work fine for western of tau and its aggregates - make sure to beep it all cold & add proteinase & phosphatase inhibitors cfr link attached 
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I extracted whole blood samples using the Qiagen DNeasy blood and tissue kit. When quantifying the DNA on the Qubit, I get baffling low DNA concentrations. Is there something that I might be doing wrong? Is there a way to increase my DNA yield after extractions?
Thank you!
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Hello  Sonja,
I have extracted a lot of fresh human blood samples using the QIAMP DNA blood mini kit from QIAGEN (cat 51106 250 samples). I use 200 -300 uL  of blood and elute in 40 uL of  buffer. The DNA concentration with Qubit   dsDNA broad range assay kit (cat Q3850 Life Techn) ranges from 40 - 400 ng/uL. When I use buffy coat also I get from 40 - 800 ng/uL depending of each sample. You need to consider a couple of things: 1. The extraction kit you use is for certain species (you do not mention blood of what species you are extracting), the genomic DNA size depend of the species and also the type of column every kit is intended for. 2. The DNA yield depends of what is the starting volume of blood you are using, you cannot overload the column.3. When you said you get a low concentration is based on your assumption or your are comparing with another DNA reading method as Nanodrop, remember the readings with QUBIT are much less that with Nanodrop (for blood is probably 3 fold less than with Nanodrop) this is because the Qubit is reading intact DNA  and it is very specific just to read good quality of DNA. I hope this help.
Cecilia Santrich
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We have since 2 Month the QX200 droplet digital PCR-equippement from BioRad. We want to introduce this as soon as possible into Routine diagnostics. as it is a new technique only Little experience can be shared. I would like to share mine. Therefore I ask here to get in contact with me to share experience in the field of Food diagnostic.
René
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We have experience/accreditation in routine food testing using qPCR and experience with BioRad's QX100 and now the QX200 for absolute quantification of DNA reference materials.
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I am detecting HPV mRNA by real time PCR. the PCR is of 50 cycles. There are many samples which show the Ct value as high as 40. Does it mean that they contain the HPV mRNA? should they be considered as a positive sample? where should I draw a line to consider them as positive or negative? 
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This is entirely dependent upon the assay: hardware platform, chemistry, cDNA input concentrations, reaction volume and Cq threshold setting; SYBRGreen based assays typically give lower Cqs than probe based (e.g. TaqMan). It is possible to derive a usable cut-off from an absolute quantification method - if you can create (or buy) an accurate control of your gene of interest (such as a plasmid quantitated by digital PCR), then by running a standard curve (typically 10^6 - 10^1 molecules per reaction volume) you can calculate an intercept, which is the hypothetical Cq for 1 molecule. Guidelines for molecular monitoring in the BCR-ABL1 setting (CML) recommend a cut-off for positivity of the Intercept Cq+1; although the quantitative range will be defined by your lowest plasmid dilution point (typically 10). (Foroni L, et al. (2011). Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia. Br J Haematol. 153:179–190). If using a SYBRGreen based relative qPCR assay, then something like 4 Cqs lower than the lowest Cq of your NTC would be a reasonable rule of thumb.
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I want to amplify the uncharacterized gene. First step, I will aplify and sequencing full cDNA of gene.
What is the best (quality and price) the RACE kit ?
(First choice, invitrogen  and   SMARTER RACE, takara  or etc.)
thank you
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Yes, it is very expensive but it is working great and no surprises but the price is here.
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Is there an agent for virus inactivation that can be used in whole blood samples, that still allows to make routine tests in those samples, like blood cell count and blood chemistry?
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Dear Enrique
There is no validated or approved technique for inactivation of viruses in whole blood, even for therapeutic use. While it is "relatively" easy to inactivate free enveloped and, to some extent as well, non-enveloped viruses in plasma, it is more complicated to inactivate cell asssociated (e.g. lymphocytes-associated) viruses without affecting the integrity of the cells. The only technique that could possibly do the job currently is photoinactivation combining a photosensitizer such as riboflavin and illumination. This technique is, however; yet not approved for clinical use and requires careful conditions for its application. Leucoreduction may remove some cell-associated viruses but would obviously affect the blood count. In conclusion, to the best of my knowledge there is no simple procedure that could ensure inactivation or removal of viruses in whole blood samples without affecting the integrity if the cells (RBC, platelets, WBC). Kind regards, Thierry
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What can be done to improve the sensitivity and specificity of the tests, aside from using proper controls?
What primers are best for Toxoplasma detection?
Real-time PCR?
Analyse cDNA to confirm the presence of living Toxoplasma - is this a viable idea?
Is the detection of antibodies considered mandatory to prove Toxoplasma infection, or will PCR results suffice if done properly? Or are there some problems that I don't know about?
Will be most grateful for any ideas on the topic.
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Hey, its all depend what is your target. For diagnostic purposes ELISA is best but some time it gives false positive results. I always prefer PCR specially nested-PCR for toxoplasma, followed by RFLP that gives fast and accurate results to identified T.gondii infection and determine the toxo genotypes
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Immunohistochemistry is a method for demonstrating the presence and location of proteins in tissue sections. Though, less sensitive quantitatively than immunoassays such as Western blotting or enzyme linked immune sorbent assay (ELISA), it allows the study of the distribution and localization of specific cellular components by providing supplemental information to the routine morphological assessment of tissues. This is especially useful for assessing the cellular markers that define specific phenotypes then providing important diagnostic, prognostic, and predictive information relative to disease status and biology. In general, the information gained from IHC combined with microscopy literally provides a “big picture” that can help make sense of data obtained using other methods.
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Immunohistochemistry confirms protein expression of selective genes in a sense that, this technique is looking at proteins. A higher gene expression in a given situation does not automatically mean that the protein is also highly expressed.
Both western blot and IHC can be used to partially validate the information given by the other technics. Both of those technics could be considered as semi quantitative as they have both potential limitations (i.e optical limitation, generated by saturation level of pixels, number of steps, amount of protein used..).
Those limitations can be solved by technical adjustments.
Once those are fixed, IHC (and WB) can be used on purpose of validating gene expression
The IHC has also the advantage of providing information on the cellular and the sub-cellular localizations of the protein of interest.
Best
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If that is possible would 2-step method more useful than 1-step?
PD is a potential by-product in PCR, a PD consists of primer molecules that have attached (hybridized) to each other, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.
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Hi
Yes primer dimers can be a problem in PCR (not only in RT-PCR) and the actual problem lies in paying less attention to the different types of primer;primer interactions during primer designing.....using unnecessary more primer conc. (than required) amy also favour such interactions during PCR..
be
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I am working in a project that l use both one step and two steps methods, but l would like to know which of them more useful and specific.
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Hi Noha,
apart from the suggestions above..
1) one step is better than 2 step for RT if you want to take care of contaminations....and it is useful when you are analysing few genes.....
2) However it offers less flexibility when it come to select the types of RT enzymes you want to use eg...if high annealing is desired for compex RNA molecules etc....you don't get both 1 and 2 step kits for all RT enzmyes
3) If you are planning analysis of laarge number of transcripts in a sample....itwould be better to do RT first and then PCR separately for different genes....many factors will be same including cDNA synthesis efficiency ...unlike doing 1step analysis for all the genes..
4) One step analysis for large number of genes will be more expensive than 2 step analysis in the same sample
5) and also ...modt commonly used 1 step RT-PCR kits have mixes with low sp. activity RT enzymes .....if you desire to have high specific activity ot RT...it may not be possible...
so decide the types of analysis and selct the methods......contamination can be taken care by a careful planning of experiments and good lab practices
bye
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Genomic imprinting differs from that of ordinary Mendelian rules of inheritance, as in the first aspect the gene is expressed by one allele from one parent and the other is silenced by imprinting; where in Mendelian the gene is expressed by two alleles coming from both parents. The imprinted genes are maintained in somatic cells, where in germline cells it is erased and re-established during development.
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Genomic imprinting is based on DNA methylation. The patterns of 5'-CpG-3' methylation dictate gene expression. Apparently, in most cases, CpG methylation is associated with gene silencing. Each individual has a pattern of DNA methylation which is established according to his/her parents' DNA methylation patterns and the methylation acquired from the environment.
Importantly, the DNA copies which are prepared in germline cells, are specifically methylated to reflect the parental methylation. This methylation appears to be produced by the maintenance DNA methyl-transferase Dnmt1.
After fertilization, the two DNA copies - maternal and paternal suffer a wave of demethylation, however, it seems that the imprinted genes (the ones known to be DNA methylated) are not affected by it. Therefore, they maintain the parental (both maternal and paternal) methylation patterns.
There are only a few genes discovered to be imprinted so far, probably about 80. A good example of such genes are H19 and Igf2 genes. You will see here that Igf2 gene imprinting is quite peculiar because expression of it is actually activated by methylation. A good paper on methylation (especially the environmental influence on methylation patterns) is the one of Waterland and Jirtle 2003.
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With the growing number of biomarkers regularly discovered, going through clinical trials and more slowly translating into the clinic, what options are available to shortlist and find biomarkers? For example for:
- Developing new diagnostics/drugs
- Researchers seeking to make new biomarker discoveries 
- Selecting biomarkers for disease diagnosis/treatment 
- other ways biomarkers are used/needed for projects
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Outliers are the goal for biomarker seekers, all the time. The key is how to find out the outliers. With the development of Bioinformation, outliers can be found more easily. However, I don't think all outliers appeared have been researched or noticed. So, reviewing results of others' research may be a way. Besides, new findinds always depent on new technology. If the current technology truly block the further finding, maybe the shortage of them should be improved.
another way is broading the area you focus, like the project, namely RIOK, started recently.http://www.r10k.org/R10K/About_R10K.html
R10K update: NGS of immune repertoire for biomarker discoveries (P3241)(J Immunol)
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I am going to generate antibodies by injecting a mixture of Freund's adjuvant and purified protein into mice. Should I use intraperitoneal injection or subcutaneous injection?
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Hello Tam,
You should take into consideration both the route of administration and volume of injection. Subcutaneous and intraperitoneal routes are most commonly used and are equally good. As you said that you are using Freund's adjuvant, if it is CFA (complete FA) then it must be limited to the initial immunizing dose. All subsequent boosters should use IFA (incomplete FA) as an adjuvant. The FA:antigen emulsified mixture of 1:1 is commonly used. Please note that intravenous (IV) administration of antigen with CFA is prohibited in mice.
Hope this will help you.
Flag this if it is really worthful.
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I am interested in the microparticles' functions in blood. I want to determine the levels of some microRNAs included microparticles by real-time qPCR. As the level of microparticles in blood is limited, I want to know if the level of the included microRNA is sufficient to be detected?
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Thank you, all. I will try it.
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A pregnant woman seeking prenatal diagnosis of beta thalassemia demonstrated the following test results:
Hemoglobin electrophoresis:
Hb A1 92.3%
Hb A2 7.7%
CBC:
Hb 11.8 g/dL
MCH 20 pg
MCV 63.6 fL
RBC 5.91 million/uL
However, when beta globin gene was sequenced, no mutations were found. The sequence information obtained covered two regions:
1. from the upstream nucleotide -101 to the nucleotide 35 of intron 2
2. from the nucleotide 556 of intron 2 till the end of 3' UTR
Only homozygous polymorphisms such as CD-2 C>T, IVS II-16 C>G, and IVS II-666 T>C were found. A definite answer is needed for genetic counseling. Any advice will be very much appreciated. Thank you.
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Patient DNA should be tested for deletion/duplication by MLPA for example. Most importantly what is the status of her partner?
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We are using F3, B3, FIP, BIP, LF and LB primers with Bst2.0 warm start polymerase (NEB), 0.8M betaine, 1x amp buffer (NEB) in 25 ul volume at 63C, 1 h, follwed by 80C, 5 min. We get good results sometimes, but nothing next time using the same reagents? Have tried to standardize by varying conc, of primers, Mg2+, etc. We isolate DNA using commercial kit and used various conc. of DNA. We aren't able to pinpoint the problem.
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Try switching to Recombinase Polymerase Amplification (RPA) instead. Reactions take <10 minutes, they can be multiplexed, real-time, end-point lateral flow, work at body temperature, are robust to off-temperature set-up, available as lyophilised reactions etc etc. Check out some publications at http://www.twistdx.co.uk/resources/publications/
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Three sets of primers, various conc. of Mg2+, 1x amp buffer (NEB), Bst2.0 warm start (NEB), 0.8M betaine, 1.4 mM each dNTPs, 25 ul finalreaction vulme, 63C, 1h, followed by 80C, 5 min. Sometimes we get expected ladder pattern of bands in AGE, next time using the same reagents, nothing. Have repeated several times- no gain.
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hello sir, I am currently doing a research on LAMP technology and was wondering if i could ask you questions regarding LAMP primer designs ? your help will be much appreciated.
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During my bibliographic research I found in Article that "Plasma ACE levels are highest in subjects with the DD genotype, thosewith the ID genotype come next, and those with the II genotype are lowest of the three genotype groups."
I would like to know if you can explain why the DD genotype is the one where the concentration of ACE is increased in plasma?
Does anyone have suggestions?
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In 'An Insertion/Deletion Polymorphism in the Angiotensin I-converting Enzyme
Gene Accounting for Half the Variance of Serum Enzyme Levels by Brigitte Rigat et al. (J. Clin. Invest. Volume 86, October 1990, 1343-1346)' it is stated that: 'the observed genetic control of serum ACE level is exerted at the transcriptional level. In that case, the insertion/deletion itself may not play a direct part in controlling ACE transcription but is more likely to be in linkage disequilibrium with regulatory elements of the ACE gene.'
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I have a problem with some sequencing results (Sanger seq., germinal DNA changes) in one exon.
I´ve obtained different results (SNP - normal allele/ heterozygote) depending on the sense of the primer. I repeated it 3-times (including new amplification) and it´s still the same. This potential change is known as nonpatogenic polymorphism, but not in Caucasians as far as I know.
Thanks.
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