- Kenneth Michael Pollard added an answer:Troubles with RNA extraction from mouse skin.I'm trying to obtain RNA from mouse skin but the results have not been satisfatory. I observed chemical contamination and also very low amounts of RNA (1-5ng). I'm working with a murine model who has collagen overproduction and usually freeze the samples directly in -80°C in RNAlater.
Qiagen support told me that the RLT buffer can crystallize and that I should use a water bath at 37°C after this specific step to dissolve the crystals (this is not recommended in the kit). Do you think this is possible even working at room temperature? Any other recommendations ?
What RIN numbers are people getting using the various protocols that have been described in this thread?Following
- Virginia Rebecca Falkenberg added an answer:What is the consensus binding motif of Dexamethasone?I want to know if Dexamethasone serves as either a transcription factor to enhance or repress transcriptional regulation.
Which bioinformatic program can I use?
You can do bioinformatic analysis using transcription factor binding site matrices to at least predict the possibility of GR-binding. However, it is the case that only an experiment, such as ChIP or EMSA, can prove the interaction is possible and a functional assay is necessary to prove the transcription factor binding can regulate transcription.
I think or that https://www.genomatix.de/ has outstanding matrices that are well curated and quite good. You can upload your sequence of interest and I think there are trial uses available for academic researchers.
You are looking for binding sites in the nuclear-receptor factor family if you are interested in dexamethasone treatment. In particular you want to look for NR3C1 (Glucocorticoid receptor) binding which are labeled as putative GRE (Glucocorticoid response elements).
If you would prefer a free program there are matrices freely accessible online in the TRANSfac database and there are numerous programs free online that use Transfac matrices to search your sequence of interest. (One example is SiteSeer). Go ahead and do at least a cursory sequence analysis before you invest in an in depth analysis. Hope this helps, and good luck!
- Sachin D Honguntikar added an answer:Can anyone help with RNA quantification before performing real time PCR?
Hello, I am extracting RNA from very few number of cells ( 500 cells) from an embryo and it cannot be detected in nanodrop, So I used same volume of RNA for both my control and test samples and synthesize cDNA. Next I amplified endogenous control and my target gene using step one real time PCR. I got result but standard error is more. So I want to know whether normalizing with endogenous control gene without quantifying RNA is acceptable method for publication?
Thank you all for your wonderful suggestions.Following
- Samah Jassam added an answer:How can I stimulate the human umbilical vein endothelial cells with histamine and TNFa within separated experiments?I need to stimulate the humvaan umbilical vein endothelial cells with Histamine and TNFa in order to see the effect of that on the barrier function of vascular endothelial cadherin catenin actin complex, so my questions:
1. How much can i use concentration in that for both Histamine and TNFa?
2. what is the time points to see after stimulation of the cells, for ex: after 1 min, 5min..etc?
3. what is the best intracellular protein that related to Vacular endothelial adherent junction and affected by Histamine and TNFa at the same time?
Please, remember, you need to keep the concentration of TNF-a close to the real level in human body, for example 12.5 Pg/mL is the concentration of TNF-a in cancer patients blood. However, to activate the expression of Slectins (CD62-E,L,P) on the endothelial cells I am using 10µg/mL. you need to apply TNF-a about 6-18 hours. I have tried this concentration and it truly worked well for me. I hope that was helpful. Good luck.Following
- Honit Piplani asked a question:Can anyone help me in designing a peptide against a specific target sequence of protein?
- Ian Teo added an answer:What are the advantages and disadvantages of using Standard curve in stead of delta delta Ct method in Real Time Gene expression assay?When we want to assess the gene expression assay by Real Time PCR, there are mainly Taqman gene expression assay or SYBR green gene expression method in which we need to set up the primer set by ourselves. By SYBR method, again there are Standard curve or delta delta Ct methods for detection of gene expression. Is there anyone who has had experience in comparing these two methods? Can you suggest which of the Standard curve or delta delta Ct method better?
I am planning to use two Endogenous controls but I don't have any positive control for this experiment. Please help me.I would always go for a standard curve method based upon diluted standards (eg plasmid ) of known concentrations with both the target gene and a reference (housekeeping) gene measured. The simple reason for this is that real copy numbers give you hard data which you can relate to your input. If I put the cDNA from the equivalent of 100,000 cells and did a PCR for a known highly expressed gene (eg ribosomal RNA) and got 100 copies, then I have reason to question my sample or cDNA. If you generate a Ct of eg 39 what does it tell you? The difference between a Ct of 45 and a Ct of 50 is the same as that between Ct 30 and Ct 35, but in terms of copy numbers a Ct of 50 in most instances is less than single copies. delta Cts and delta-delta Cts are fast , easy and lazy ways of getting data. When analysing someone else's data, unless you know the range of Cts from which the delta Cts were obtained it is difficult to determine whether the data is truly within the range of believable results.(someone once presented me a graph of a 30 fold difference based a upon a Ct difference of 50-55 cycles ). A value of 1000 copies of x per million copies of ribosomal RNA is a hard number.These real values can then be used to determine the fold change in expression. It also makes direct comparison between experiments more realistic.
Remember: High impact factor = High input effortFollowing
- Yasser Cabansag added an answer:Can anyone help me regarding real-time PCR using the sybr green method?I am looking for gene expression by using the sybr green method. In my melt curve analysis, I am getting multiple tm peaks for target gene, whereas my endogenous control gene melt curve is perfect. However when I checked the PCR product of my target gene on the gel, I am getting single band. Can anyone suggest how to overcome this problem?I think your control is more optimized on your thermal profile compare to your target. Multiple peaks sometimes suggest that you did not get the right annealing temperature for your primer. Also, qPCR is more sensitive compare to conventional PCR. Even a very small amount of DNA can be amplified. Maybe the peaks are too small in amount that you can not able to see them in gel, that is why you can only see single bands.Following
- Mohamed Samir added an answer:Why are my no template controls for qPCR yielding products?My "no template" controls are showing products that are identical to their genes. I did bax and p53 qPCR and noticed that bax amplification was almost identical to bax no template control; p53, the same. Their Cq values were also identical. Their melting curves also looked the same. A single peak was shown on their melting curve. Is the problem with my primers? What other issues could cause this? DNA contamination of reagents?You may try to use less concentration of primers. make several dilution and try it all. I expect that you will get no signals in a lower primer concentration.Following
- Azeem Mehmood Butt asked a question:Has anyone used the spike-in for circulating microRNA?I would like to know if there is anyone who has worked specifically with QIAGEN cel-miR 39 spike-in using the QIAGEN miRneasy mini kit.Following
- Martin Devonshire added an answer:How can I get rid of a non-specific binding with GST- beads?I am doing protein mapping to my interesting protein (chop it off for different domains, mix them with other GST- protein to do in vitro binding assay and then they are pulled down with GST- beads), but I realized that some domains bound to GST-beads in the controls. Is there any suggestion?you cannot.... you will be able to minimise it somewhat but u would be amazed if you got rid of it , i would do the best you can with varying salt concentration and detergent, maybe try changing buffering (Hepes instead of tris for example) then when you have the prep as good as you can get it i remove the GST (if that is desirable) then use a size exclusion column to further purify the protein of interestFollowing
- Anirban Chatterjee added an answer:Can anyone explain variable results from Ct values analysis?I would like to do relative quantification of my real time PCR data, and I have designed a program based on the literature calculation and other ready programs, but the results appear variable, sometimes high or low. My sample FFPE.Dear Dhafer,
I suspect that all your FFPE samples are not uniform in their tissue type and content. What I meant to say that they might be containing variable amount of utilizable RNA. And secondly you might be be having some minute differences with your RNA isolations from your FFPE samples, which are then getting magnified in qRT-PCR.
You can go for the following Kit from Roche, it usually gives a steady result with efficient yield:Following
- K.s Sim added an answer:How to use RNALater with bacterial liquid cultures?Is it possible to add RNALater directly to bacterial cultures instead of centrifuging first and then resuspending them in RNALater? If yes, then what should be the ratio of cell suspension:RNALater?Peter Kos,
may I know is there any publication regard Phenol-EtOH ?
while, may I know the % of ice cold ethanol ? can ethanol be replace with isopropanol ?
- Jochen Wilhelm added an answer:Which is the best choice to determinate qPCR amplification efficiency? cDNA, gDNA or plasmid serial dilutions?Dear colleagues
I'm working with qPCR relative quantification and I need to determine the efficiency of qPCR amplification. I know we can use dilution series from gDNA, cDNA or template inside a plasmid, but what are the advantages and drawbacks of each one? what is the best choice?
I would really appreciate if you can guide me
Thanks in advance
My best regardsThe strands of denatured plasmids will not move apart, they are interwoven. Therefore their re-hybridization(naturation) won't follow a first-order but a zero-order kinetic of a very high rate. This can easily interfere (compete) with primer-annealing. Also, stand-displacement by the polymerase in a plasmid is sterically more difficult. This can result in a lower amplification efficiency.
Regarding Stephens comment: contamination is an important issue, no doubts that one should take as much care as possible. However, If the standard preparation is done in a different lab (best in a different building!) and only highly diluted "working aliquots" are taken to the lab where the PCRs are pipetted, a contamination risk is *relaively* low (given recommended standards in clean and careful working).
The standards should not be taken "as is", they should be spiked to "real samples" with no or negligible own amount of the target sequence.Following
- Noha Shendy added an answer:How can I isolate WBC for RNA extraction?Can anyone please share a protocol for isolation of white blood cells and extraction of RNA from these cells (RNA isolation from these cells only)?Following
- Andrew J Saurin added an answer:Is there any databases to get primers for ChIP analysis?.And for anything not in a db or published, I find NCBIs primer-blast very useful for designing specific primer:
- Zuly Pava added an answer:Has anyone tried to determine primers efficiency using total RNA in a one-step RTq-PCR (SYBRgreen) reaction?I've tried to test my primers efficiency by using a one-step rtq-PCR (SYBRgreen) with serial dilutions of total RNA and using RotorGene software. I've got the same result as reported in the literature: efficiencies higher than 100%. So, I'm wondering if there's an alternative other than plasmids, to carry on this experiments. I'd appreciate in advance ANY suggestion.Thanks a lot, the cDNA serial dilutions worked much better.Following
- Tobias Meißner added an answer:Software for Affymetrix miRNA 3.0 array data analysis? What is advantage of using Gene spring and what are limitations of expression console?I am learning Affymetrix miRNA 3.0 array data analysis? I want to know the difference between Gene spring and expression console? How do they differ in statistical analysis?Following
- Tushar Tomar added an answer:How can I incorporate fold change value in heatmap on untreated condition?e.g. Gene-A has a normalized value in untreated condition 200 and drug-treated condition 20, so the log2(ratio) is -3.32. On the other hand, Gene-B has a normalized value in untreated condition 20 and drug-treated condition, 200, so the log2(ratio) is 3.32. If I label the upregulation (red) downregulation (green), I can get the heatmap on treated sample (3.32 fold up or downregulation), but what would be the fold change value in untreated condition?I completely agree with Jochen in this matter. However, sometime I found that people use log2 transformed of fold induction and present control also as 0 (Log2 (1)) in the heat map. SO black color for control and red/green for treated group....but I personally don´t think its a good way of representation.Following
- Kishna Vijayaraghavan added an answer:How do I control noise expression in genetic switches?Does anyone know if there is any experimental system for study noise minimisation in gene expression? I trying to verify the predictions done in: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0084020You pls refer this link : http://www.pnas.org/content/97/5/2075.full.pdfFollowing
- Khalid Beshir added an answer:Optimal primer concentration for real time PCR?Can anyone please tell working concentration of primers (in nanograms/picograms) for real time PCR experiment. Because I have primers stock at 2 microgram concentration.Jack has clearly answered your question. However, you will have to try different concentrations (in addition to 300nM final conc as suggested by Jack and Jonathan) and choose the lowest concentration that gives you good result.Following
- Tushar Tomar added an answer:Need help with Agilent miRNA microarray data analysis.I've used the "AgiMicroRna" package of 'bioconductor' using R to analyze my miRNA microarray data. till data analysis was just fine. Arriving at diffferential expression was butter smooth using Pedro Lopez's guide to AgiMicrRna package. Now further on to gene annotation, pathway enrichment GO and interactome (KEGG).etc. lies the hurdle.. I'd really appreciate inputs from one and all in this regard...Any body done this before..??..could you share your strings with me..??Generally uses: TargetSCAN for miR gene annotations and then DAVID for functional clustering and annotations
- Sachin D Honguntikar added an answer:Can anyone help with quantification of cDNA in control and test samples?I have isolated RNA from few cells (700-800), which cannot be quantified by nanodrop. So can I synthesize cDNA taking same volume of RNA from ctrl and test group and quantify the cDNA and use same concentration of cDNA ( both ctrl and test) for real time PCR (SYBR green method)? And how can I determine how much fold of change in gene expression in ctrl and test?Thank you all for your suggestions. I will normalize with any of house keeping genes.Following
- Satish kumar Noonepalle added an answer:How to identify transcription factors binding to a specific DNA sequence?I have an idea to identify the transcription factors binding to a specific DNA region. I don't have any transcription factor candidate, only what I have is a potential promoter region of a gene. Does anyone have any idea what I should do, what techniques should I use? or any software to tell the transcription factor and gene binding?Use ENCODE. Its very helpfulFollowing
- Ales Vicha added an answer:Does anybody have rest 2009 software?Statiscally analyze gene expression result.Thank you very much to help me. I also, installed Windows XP Mode on my 64-bit Windows 7. Additionally it requires NET framework 2.0, make sure you install 32-bit version. Now, it worksFollowing
- Karol Szafranski added an answer:Is a 2x50 bp paired end sequencing read sufficient to identify differentially expressed transcripts by Illumina RNA-seq?I'm gearing up to do an RNA-seq experiment using the Illumina platform to identify differentially expressed transcripts in liver RNA samples from a mouse study. The output would be a minimum 30 million read depth and paired end sequences (i.e. from both ends of each transcript). I'm trying to determine if 2x75 bp reads is worth the extra cost, or if 2x50 bp sequences (i.e. 100 bp total per RNA) would be sufficient to align and identify unique transcripts. I'd appreciate any advice or experience.1x50 nt reads, i.e. single-sided sequences, are sufficient for a standard expression analysis in a model organism. Longer read length or paired reads I would recommend only if you aim at complicated genes, like paralogous gene families with high sequence similarity, or at detailed genes structures, like certain mRNA isoforms (alternative splicing). This recommendation is in accord to the ENCODE guidlines cited by Aureliano.
One thing that may be important for your considerations: What mouse strain do you want to analyze? If it is very different from C57Bl/6, then your read mapping may become affected. And this might be a reason to increase the read length (possibly 75 nt) in order to keep your mappings specific.Following
- Rohan Chaubal added an answer:Normalization to average as an alternative to using reference gene.Is it valid to normalize gene expression to the average between biological repeats? Could that approach be used for large data sets were most genes show some degree of variation or is there a strict requirement for a reference gene?Following
About Quantitative Gene Regulation
A group for scientists interested in quantitative descriptions of gene regulation in pro- and eukaryotes (equilibrium and non-equilibrium protein binding, chromatin rearrangements, covalent modifications, input-output cis-regulatory functions, etc) using approaches of biophysics, molecular and cell biology, bioinformatics, systems biology and synthetic biology.