I am currently working to design several primers for PCR (RT-qPCR) and would like to validate them. As of now, I am using Primer-BLAST, both to check for my primers and some from the litterature. If some come back as targeting a completely different gene than what was expected, can I consider the results as final?
In simple without indulging into deep science and molecules bizarre behaviors and to save time just run your designed primers into qPCR by using control cDNA in 1 uL (usually provided by the companies and standard one is 25 ng / uL), use primers in various concentrations (like 2, 5 and 10 pmoles / uL) by qPCR master mix (like Sybr Green) and run each concentration in triplicates at least. And check the amplification plots and melt curve peaks. In your same qPCR experiment also perform control which is without sample and use only primer & master mix to get an estimation about the primer dimers and their Tm on melt curve plot so you can compare your product melt curves with these temperatures which will be quite lower than the products. Then get this product out from corresponding wells and run on Agarose gel (2.5 %) and compare the size of your product. Although it is often indicated that qPCR products are best viewed on 6 - 19 % Acrylamide gels, but Agarose is fine too. On the other hand, we can also try these primers with any conventional PCR master mix by using the same control cDNA and same run protocol of qPCR builtin program in conventional thermal cycler, and then run on gel and check the band sizes. If primers are good so precise product bands are there. This will give you good peace of mind...
No~! eek gaads. ;]
The algorithms you employ for primer design are only as valid as the final mastermix you use. Every mastermix has its own personality. The theory of Tm algorithms is only as good as the final environment to which the resulting oligos are entrusted.
(The Beatles: the love you take is equal to the love you make).
Once you design primers, you must then choose a mastermix, after that, you must witness how everything behaves in your own hands and so on.
Tm algorithms are merely best-guess suggestions. Ionic strength and primer concentrations both impact effective Tm of primers in solution. This is just the tip of the iceberg. Nothing is really "final" in these matters. Dig as deep as you can before you proceed. And realize, there is much mis-information out there. You cannot mine the sequence databases "too much." Go slowly and carefully through these things before you use the word "final." That is my opinion anyway.
In simple without indulging into deep science and molecules bizarre behaviors and to save time just run your designed primers into qPCR by using control cDNA in 1 uL (usually provided by the companies and standard one is 25 ng / uL), use primers in various concentrations (like 2, 5 and 10 pmoles / uL) by qPCR master mix (like Sybr Green) and run each concentration in triplicates at least. And check the amplification plots and melt curve peaks. In your same qPCR experiment also perform control which is without sample and use only primer & master mix to get an estimation about the primer dimers and their Tm on melt curve plot so you can compare your product melt curves with these temperatures which will be quite lower than the products. Then get this product out from corresponding wells and run on Agarose gel (2.5 %) and compare the size of your product. Although it is often indicated that qPCR products are best viewed on 6 - 19 % Acrylamide gels, but Agarose is fine too. On the other hand, we can also try these primers with any conventional PCR master mix by using the same control cDNA and same run protocol of qPCR builtin program in conventional thermal cycler, and then run on gel and check the band sizes. If primers are good so precise product bands are there. This will give you good peace of mind...
Dear Luc
I recommand you some manuscript:
1-Guidelines for validation of qualitative real-time PCR methods. Broeders et al. Trends in Food Science & Technology . Volume 37, Issue 2, June 2014, Pages 115–126. Available at: http://www.sciencedirect.com/science/article/pii/S0924224414000661
Abstract: As for many areas of molecular testing, detection of Genetically Modified Organisms (GMO) relies on the real-time Polymerase Chain Reaction (qPCR) technology. Due to the increasing number of GMO, a screening approach using qualitative screening methods has become an integrated part of GMO detection. However, specific guidelines for the validation of these methods are lacking. Here, a pragmatic approach to conduct in-house and inter-laboratory validation studies for GMO screening methods, is proposed. Such guidelines could be adapted to other areas where qualitative qPCR methods are used for molecular testing allowing to implement easily a more reliable screening phase where necessary.
2-Designing & Validating Real-Time PCR Primers: Systematic Guidelines available at the following link: http://www.sabiosciences.com/newsletter/PATHWAYS07_PCRprimers.pdf
Hoping it will be helpful
Marius
- Badges
- Science method