It was a pleasure meeting and working with you all today and I hope you found the seminar informative and useful. Any feedback is welcome!
Please forward this E-mail to all your colleagues interested in qPCR. Please read “The Practical Approach to qPCR…” and the “MIQE Case Study…” papers which are attached to this message before embarking on your experiments. Both papers would make good journal club or lab talk topics. If you have limited time, we have very recently published a short article that describes the key steps in RT-qPCR experimental design according to the MIQE Guidelines which can be downloaded at the following link ( http://bit.ly/JMMB_Taylor ) (http://www.karger.com/Article/FullText/356189 ).
I have attached the two tables from this article entitled the Do’s and Don’ts of qPCR. My suggestions for reagents are summarized as follows:
qPCR reagent recommendations:
1. RNA Isolation: The Bio-Rad Aurum kits are excellent and typically yield nice, clean RNA preps for the downstream work. However, if you are working with very small tissue samples (<25mg), the Quiagen kits give good results. If you are doing mammalian cell culture or isolating mammalian cells from serum with very small numbers of cells use the iScript™ RT-qPCR Sample Preparation Reagent with the iScript™ One-Step RT-PCR Kit with SYBR Green (or for probes). For plant material, there are a number of kits and home brew methods available but always validate your primers with the resulting gDNA or cDNA with a thermal gradient and a standard curve using a pooled sample to be sure that the resulting sample is OK for qPCR.
2. cDNA Synthesis: Any of our iScript kits are strongly recommended for their coverage of the transcriptome, the fidelity of the enzyme and the quality of the downstream qPCR reactions in terms of sensitivity and reproducibility. I have found that folks who try these kits are very impressed and they are typically less expensive than those from other companies.
3. qPCR: I am a big fan of our SsoAdvanced™ SYBR® Green, EvaGreen or Probes Supermixes. The main reason is that the enzyme in these mixes is a fusion protein of Taq and the Sso7d protein which finds and binds to the DNA with a much higher binding capacity than the Taq in other commercial mixes. This makes all the Sso mixes much more resistant to potential qPCR inhibitors that can contaminate the RNA and associated cDNA providing more reproducible results.
4. PrimePCR Assays: This is our solution for those who wish to move faster from experiment to data. We have the full human and mouse genomes of pre-designed and fully validated PCR primer pairs for use with dye-based chemistry such as SYBR® Green or EvaGreen®. The primers can be ordered individually in tubes or pre-plated on 96-well plates to test with your samples. This is an excellent option to screen may genes from a pathway or disease panel or for reference genes (http://www.bio-rad.com/en-us/product/pcr-primers-probes-panels).
Feel free to contact Nadine Menhem (cc’d on this message) if you would like more details. Here are the links to download CFX Manager 3.1 and some training videos I recorded for qPCR and ddPCR:
CFX Manager 3.1 Software: https://app.box.com/s/3bgzwdolr63w6n8aoawo
Let me know if you have further questions and good luck with your research in which Bio-Rad can play an important role!
Field Application Specialist
For All Technical Support