if the primers have been used in other papers and you want to cite these primers in your paper for relative quantitative real time PCR, do you have to do the standard curve and calculate the amplification efficiency?
Laurence Stuart Dawkins-Hall
Dear Liang
- In short no you do not
- Nevertheless, if you can perform a standard curve then it is still advisable to do so
- That is because knowing the amplification efficiency is useful when it comes to relative quantification of transcripts in test samples versus controls; either in order to perform the Livak (delta delta Ct method) of gene relative quantification which utilises a co efficient of '2' and thus presupposes a perfect doubling during the logarithmic phase of your qPCR amplification curve (for that to hold you ideally should have primers with amplification efficiencies > 85%) or the Pfaffl method which corrects for primers with sub optimal efficiency ( 75%-85%) by inclusion of the actual efficiency, supplied by the standard curve
- In addition, the standard curve will tell you the lowest dilution of cDNA compatible with Ct(p) values between + 15-+25/30 cycles which is the ambient Ct range
- I say lowest dilution because higher concentrations culminating in Ct values < 20 can encourage non specific products/primer dimers in some (but not all) instances
- Nevertheless, for some low copy number genes yielding Ct values > 30 it is not (without pre amplification) practicable to perform standard curves: In these instances providing you can demonstrate that Ct values from triplicate (technical) replicates are
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Liang Yusheng
Dear Dr. Laurence:
Thank you very much, your suggestion is very useful.
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Liang Yusheng
Dear Dr. Laurence:
If convenient, can you send me some materials which are related with this question. My e-mail address is [email protected]. Thank you very much.
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Laurence Stuart Dawkins-Hall
Dear Liang
- I enclose a pdf of the stratagene general guide: This is the best general guide I have come across and addresses your issues including a bibliography referring to matters such as your own
- In addition find a paper and how and why primer efficiency estimates are important
- I have included a second publication on sources of error in qPCR calculations including differences in PCR efficiency between sample and housekeeping samples: In essence, both sets of primers for Livak (delta delta Ct) should have primer efficiencies > 80% and regarding housekeeping and GOI primers they should ideally be within 5% efficiency value of one another : That said this is the ideal and providing the difference in expression between treated and untreated samples is orders of magnitude, i.e. > 2.0 and certainly if > (say) 5 x then large disparities in primer efficiency between treated and untreated samples will not affect the data trend, i.e. whether the difference is statistically significant or not @ P 5% if if your primer efficiencies fall between 75% and 80% you can always account for those differences using the Pfaffl equation instead of the Livak method; To that end find attached the paper by Pfaffl. Also:
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695/
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