- Kenn Gerdes added an answer:How stable is RNAstable?
We've tried several times to send RNA samples in RNA stable but with very bad results. Initially we dried samples in laminar hood. It took more than overnight, the samples did not look completely dry and bioanalyzer results were really bad. We then tried drying in a speedvac, which took about one hour, again some samples did not look entirely dry and bioanalyzer results were not sufficiently good. We thought that our mistake was that we didn't pre-run the speedvac. So, the third time we did that, samples were completely dry in about 20 minutes, and bioanalyzer results were better. Last time we did everything "right" but bioanalyzer results were very bad again. Any ideas?
You should phenol extract your sample as quickly as possible after harvesting your cells
- Herbert Auer added an answer:How to test the RNA integrity?
Is there any other ways of testing the RNA integrity without using bioanalyzer or denaturing gel?
Has anyone tried bleach gel before? I tried it but it doesn't work well.
Wondering if anyone is able to help me troubleshoot
Why do you not want to use a Bioanalyzer or Experion or a similar instrument? This is the gold standard and there are plenty of service providers that you can send the samples to. One of them is Micromon at your university.Following
- John Hildyard added an answer:What can I do to make my qPCR results "solid"?
Hi everyone, I am working on detecting expression levels of genes among sorted tumour samples (by FACS).
I sorted the 1st tumour into PBS. Bioanalyzer showed to me really bad RIN numbers in these 4 RNA samples extracted from sorted fractions. However, I still used them to run a qPCR (Taqman gene expression assay). I got expected results in all 15 selected genes.
After that, I started working on optimising method/protocol to improve sample RIN by using cultured human cancer cell line. 2 months later, I could consistently get RNA samples with RINs between 7.5-9 from sorted cancer cells. Then, i went back to human tumor xenografts. Samples prepared from both the 2nd and 3rd tumours demonstrated very similar story in the expression all these 15 genes. So far, qPCR results from 3 tumours showed me quite constant results in all selected 15 genes.
However, there were no RNA could be detected by Bioanalyzer in all samples prepared from both tumour No.2 and 3. Today, I re-checked these samples by Qubit. It gave me completely different results comparing to NanoDrop. RNA could not be detected in all samples by Qubit either.
I am really wondering how come i can get consistent qPCR results from samples either with bad RIN numbers or with undetectable RNA levels??? (I setup qPCRs by using sample RNA concentrations provided by NanoDrop).
If all results I got came from some kind of random amplification, how come it consistent in all 15 genes? If these qPCR results are usable, what can I do to make these qPCR results solid?
Thank you very much! I look forward any suggestions/advices!!! Thank you :)
How much RNA are you using to prepare cDNA? (ballpark figure is fine)
How much of this cDNA are you using per well in qPCR?
What sort of Cq values are you obtaining?
Are you including no template controls?
Are you perfoming melt curves?
Have you checked your PCR products are of the correct size (say, via gel)?
Have you generated a standard curve?
How are you normalising your data?
Answers to all of these questions would help identify whether there are any problems with your experiments.
When performed correctly, qPCR can be enormously useful, but there are a lot of potential pitfalls to be wary of (and frankly, my first response to getting "exactly the result I was expecting" would be one of extreme suspicion, though I may be getting cynical).Following
- Parul Agarwal asked a question:Total RNA extraction?I am extracting total RNA from leaf samples using Purelink Plant RNA Reagent, followed by DNAse treatment and RNA clean-up according to Qiagen RNeasy kit protocol. On analysing my samples on bioanalyzer, the RIN is more than 7 and I get sharp peaks for 18s and 28s, but peak for 18s is higher than for 28s, 28s/18s ratio is less than 1.0. Has anyone else faced the same issue and any suggestions about how to get over this? Or would this sample be good to make cDNA?Following
- Rutendo Kupara added an answer:How can I homogenize cardiac tissue?I'm trying to develop a method for quantification of drugs (small molecules) in cardiac tissue with LC/MS-MS. The first step is homogenization with ultrapure water with a gentleMAC dissociator (rotor-stator homogenization) followed by a protein precipitation (PP). However, I'm in doubt whether the homogenization and PP adequately disrupts the cells, so I am measuring the total concentration of the drugs. Do anyone have any experience with quantification of drugs in cardiac tissue (or similar tissue)?Im using liquid nitrogen to crush my samples, works perfectly and its quick and easy, however what buffers can you help by telling me what buffers I can use to homogenize my samples which preserve the ezyme activity in the samples?Following
- Abhinay Ramaprasad added an answer:Assessing integrity of plant RNA with Agilent 2100 Bioanalyzer: What is the meaning of RIN = N/A?Quality control tests of total RNA of some of my plant samples revealed no RIN (RNA integrity number) values, i.e. "N/A", while other samples got good RINs (between 8.9-10). The RNA concentrations were between 331-487 ng/µl (measured via NanoDrop and Qubit). What's behind a 'no' RIN value?The Bioanalyser software sometimes makes mistake in recognizing the two rRNA bands either because of very low/high concentration of input RNA or if your samples has many other RNA peaks (not necessarily degraded RNA). This causes it to miscalculate or report RIN as N/A. Check if you are loading within the RNA concentration range as specified in the kit. But I would rather go by visual inspection than the RIN number. If you are getting two neat rRNA peaks ( you could also compare this with the samples where you got RIN 8.9 and 10) and low MW peak of degraded RNA (if any) of very low concentration, you are good to go.Following
- Paola Ibelles added an answer:Can you help me with identification of proteins in HDL?I have obtained by ultracentrifugation a band of HDL, mainly HDL3 characteristics, with a protein concentration of 10g / l. Can anyone advise me on the steps of how to identify these proteins?Some strategie for characterization could be the chemical and protein composition and the size distribution. You can try this approach with the methodology described in this paper. There is also a lot of methodologys for to analyze their functions as antioaxidant, at the cholesterol metabolism an more. Is up to what you are more interested on the papers that you could read. Decreased activity of lecithin:cholesterol acyltransferase and hepatic lipase in chronic hypothyroid rats: Implications for reverse cholesterol transport. Molecular and Cellular Biochemistry 246: 51–56, 2003. Palmitic acid in HDL is associated to low apo A-I fractional catabolic rates in vivo. Clinica Chimica Acta 378 (2007) 53 –58 Enzymatic assessment of cholesterol on electrophoresis gels for estimating HDL size distribution and plasma concentrations of HDL subclasses. Journal of Lipid Research Volume 51, 2010 Good luckFollowing
- Gellert Karvaly added an answer:What's the optimal approach to assaying 25-hydroxyvitamin D2 and 25-hydroxyvitami D3 in human blood by HPLC-UV?I have found HPLC applications employing normal as well as reversed phase separations for monitoring the blood levels of 25OHD2 and 25OHD3. I wonder what the routine users of such D-vitamin assays think is the optimal approach to developing a suitable method. In addition, I would appreciate having the opinion of colleagues who use commercial HPLC in vitro diagnostic kits for this purpose on the utility and potential disadvantages of these kits.Thank you indeed for your comment. I am aware that LCMS would be the first choice but unfortunately it won't be available for this project. There had been lots of publications describing the use of UV detection prior to the LCMS era, though. I expect some people had been using some of those stuffs and can share their experience.Following
- Krishna rao Dara added an answer:How can you quantify doxorubacin liposomal formulation by using LCMS/MS?Extraction procedure for liposomal doxorubacin, total doxorubacin,normal doxorubacin and its estimation.The separation was based upon the selective retention of liposomal (encapsulated) doxorubicin and non-liposomal (free) doxorubicin on the hydrophobic reversed-phase Thermo Scientific™ SOLA™ HRP solid phase extraction (SPE) cartridge. The former exhibited no retention, while the latter was retained on the stationary phase and eluted with acidified methanol.After separation, each individual fraction was quantified by UV (254 nm) also possible. You can contact me further details on +91-8980045413.Following
- Dan Brian Short added an answer:Can anybody recommend a kit to measure calcium ion concentration in solution?I am currently looking for a kit to determine calcium concentration in solution (not intracellular). Test should work in the 10 - 1000 mg/L range. Any suggestions?Hach makes these. http://www.hach.com/Following
- Edward Russak added an answer:Can someone tell me about human fluorosis cases in Sindh?I am working on fluoride dangers in humans. I need information on Fluoride-related medical cases in Pakistan, especially in Sindh.Have you read: Research report Fluoride 45(4)384–388 October-December 2012Following
- Mohamed Samir added an answer:What might be the cause of bad quality RIN in Bioanalyzer?Could anyone give me help. I sent a homogenized sample from ducks to a Bioanalyzer unite to make a quality assurance test prior to small RNA seq. Some of the samples came without RIN. Some of them show a smeared Band. Others with a RIN of 2.3. Actually I sent 300ng/µl of RNA and the person in the unite diluted the sample 1:10 and he used a Pico chip for the analysis. What might be the cause of this? Do you think that this results can be suitable for small RNA seq knowing that these small RNAs are more stable.Thanks to all of youFollowing
- Can Quan added an answer:What data do I require to measure the Uncertainty of purity for CRM?Can someone guide me thoroughly?This article might be helpful for you : There're many ariticles: http://jat.oxfordjournals.org/content/33/8/532.full.pdfFollowing
- Jean Rene Grezes added an answer:Does anyone know a suitable calcite/CaCO3 fluorescent marker?I want to dye/tag calcite particles. Anyone experienced with calcein in eukaryotic cell cultures? Any other suggestions?I agree with the answers of the 5 researchers concerning the choice of a marker for calcite particles. But if you want a concrete help concerning your research, you must be give more infos when you write : " I want to dye/Tag calcite particles...." in eukariotic cells. At first, you must give infos about the kind of your eukar. cells and the growing medium used. The organisms of various eukar. cells are able to produce biomaterials like CaCo3 e.g. calcite in various shapes ( intra and later extra-cellulars. On other part, the pH of your medium is important for the production of the Ca bio-particles. I join 2 links of different researchers Ca researchers: Author: Jason W. Rosch et al -MolecularMicrobiology (2008) 70 (2), 435–444 doi:10.1111/j.1365-2958.2008.06425.x First published online 10 September 2008 and Author : A. Scheffel http://www.mpimp-golm.mpg.de/15419/2scheffel At last, The homepage of Molecular Probes gives a lot of infos concerning Calcein AM and related Ca markers. Please give more infos the next time. I wish you a successful research. J.R. G.Following
- Denis Baev added an answer:Benchtop Flow-Cytometer upgrade - can anyone help?Is it possible to upgrade a benchtop flow-cytometer like guava easy cyte to detect orthogonal and polarized side scatter? Or is it impossible with this device?No, the simple upgrade is not possible for such type of instruments (with fixed optical scheme).Following
- Jean-Baptiste Cazier added an answer:Renaming BioInformatics?Following the "successful" debate on the definition of bioinformatics and its elusive scope. It might be time to look for a more appropriate name to cover the many forms it takes. So far, none of its subgroups, such as computational biology, data mining, mathematical modelling or biostatistics, manages to encompass its entirety. Should we coin a new term that relies less heavily on the "informatics", which is now more a side tool than the real expertise of most bioinformaticians ? The first one coming to mind would be "bio-Analytics" as all this is essentially the analysis of biological data. Unfortunately this term seems to be already taken by a rather different field. What would you suggest?Dear M Hofri, The problem lies in the perception of what Bioinformatic is: Unlike food chemist, physical chemist, etc. there is no accepted common umbrella. Most computational biologists, System biologists, Biostatisticians, etc will not agree to be labelled as Bioinformatician. However someone peripheral to the field(s) will indeed put any of them under the same banner which is not representative. The question is to find a name that would satisfy most of computational biologists, System biologists, Biostatisticians, Bioinformaticians, etc.Following
- Ragni Vora added an answer:I am making a complete body calculator online, if you were one of my customers online what would you like to see or calculate for yourself?I want it to be a complete one stop solution for health for all online customers.Yes that can be done....depending on various body factors i can explain the risk of various disease that u might haveFollowing
- Jack Silver added an answer:Acetone as an alternate solvent to acetonitrile for small molecule analysis by LC-MS/MSAs you are all aware of the global shortage of acetonitrile a few years ago, i felt it is the high time for everyone of us to look for alternate solvents like methanol or acetone for SMALL MOLECULE ANALYSIS by liquid chromatography tandem mass spectrometry to prevent such shortages ahead. Apart from methanol, acetone possibly could be a suitable alternate solvent for LC-MS/MS analysis. i have read very few articles in which they replaced acetonitrile with acetone for LARGE MOLECULE ANALYSIS by LC-MS/MS. we are planning to develop bioanalytical methods using acetone for small molecules although acetone is non-polar, which is not suitable for RP-HPLC and highly inflammable solvent (LC-MS/MS requires the application of different cone voltages) compared to acetonitrile. i would like to know whether anyone have ever tried acetone for small molecule analysis by RP-HPLC-MS/MS or just share your inputs on this.I've used acetone for PTFE (Teflon) and PEEK fittings with no difficulties. Best to contact your HPLC manufacturer since the know what materials they used. You can also look up the compatibility if you have access to a Compass guide (lists solvent compatibility of a large number of plastics and metals and solvents), if you know what they use for polymers. Here's a link to some solvent/polymer compatibility (click the polymer on the right side): http://www.zeusinc.com/technicalservices/technicalbulletins/chemicalresistanceofpolymers.aspxFollowing
- Alaa Khedr added an answer:Where can I find a comprehensive database/bank on spectroscopic information of bio-molecules?Any database/ bank on UV-VIS spectra of the biomoleculesThank youFollowing
- Prasanna Raja asked a question:Can anybody tell me about the methods available for the confirmation of carbohydrates?What are the techniques available for the separation of carbohydrates?Following
- Martin Müller added an answer:What are the analytic methods for collagens?What are all the ways that collagen can be characterized - bioanalytical tools, application studies and biomaterial studies? Kindly suggest the methods and the tools.Here is also one from our institute based on CD-Spectroscopy. Best regards, Martin Müller U. Freudenberg, S.H. Behrens, P.B. Welzel, M. Müller, M. Grimmer, K. Salchert, T. Taeger, K. Schmidt, W. Pompe, C. Werner, Electrostatic interactions modulate the conformation of collagen I, Biophysical Journal, 92, 2108-2119 (2007)Following
- Closed account added an answer:How to get amlodipine from amlodipine besylate?Amlodipine tablets contains amlodipine besylate, how can they be separated from each other?By simply extraction processFollowing
- Rajendra Bhadane added an answer:How can we identify presence of diclofenac in Biological Product..?How can we identify presence of diclofenac in Biological Product..?actually it is quite difficult to work with this type of method on UV. As there is problem with cut off wavelength and matrix effect. even there is problem with detection limit in UV. As your samples are in nano gram range which you can not detect in UV. but if you interested then you first develop the method on UV. see the linearity and range on UV by preparing multiple dilutions of diffrect conc.Following
- Praveen Kumar Vittala added an answer:Bioanalytical Method Development of NicotineHas anyone done bioanalytical method development of nicotine and its metabolites in plasma by LC-MS/MS? I am facing peak shape problems and interference problems, can anyone suggest some methods?For analysis of Nocitine, the main problem would be interfernce and matrix effect, working with SPE using Cation exchange chemistry has found to be good option for the drug and metabolites with recovery >90%Following
- Shafaq Noori added an answer:Does anyone know what solutions Docetaxol is both soluble and stable?I am working with Docetaxol and it doesn't appear to be stable in solutions that it is soluble in. Does anyone have any experience making Docetaxol solutions?5 % DMSO and acetontrile , 1-4 % DMSO check at different percentagesFollowing
Estimation of drugs in biological fluids