DNA as a standard for absolute quantitation - (May/06/2005 )
Why can't we use DNA as a standard for absolute quantitation of
RNA in RT-qPCR? 
Hope for replies.
Thanks a bunch!
Dear indoubt,
Of cause you can't. Think logically, your RNA is single stranded but DNA is double stranded.
single copy of dsDNA molecule will yield 2 copies of dsDNA after 1 cycle of PCR.
However 1 copy of cDNA (generated from RT step) will only become 1 copy of dsDNA odter 1 cycle of PCR. Due to the unequal of the starting material, the different of PCR product generated will be very great after many cycles.
Thus RNA standard must be used in RT-PCR.
I hope my answer to not too late for you.
Best regards
Of cause you can't. Think logically, your RNA is single stranded but DNA is double stranded.
single copy of dsDNA molecule will yield 2 copies of dsDNA after 1 cycle of PCR.
However 1 copy of cDNA (generated from RT step) will only become 1 copy of dsDNA odter 1 cycle of PCR. Due to the unequal of the starting material, the different of PCR product generated will be very great after many cycles.
Thus RNA standard must be used in RT-PCR.
I hope my answer to not too late for you.
Best regards
Hello,
I know this may be silly but what if you divided the copy number obtained from a DNA template by 2 and relate it to the number for RNA.
After one cycle, the cDNA product is now double stranded. It will act just the same as any other piece of dsDNA in the PCR reaction. So, I see no reason that DNA cannot be used as a standard, assuming you do not need to quantify the performance of the reverse transcriptase reaction -- but this is a major, major issue, which may be very important or dominant.
According to ABI's User Bulletin #2, "DNA cannot be used as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription step."
However, you can use DNA for the relative quantitation of RNA, and assume that the RT efficiency of the target is the same in all samples.
Good luck!
that is of course preuming you know exactly how many copies of both the gene and the expressed mRNA are present in each sample
You can use DNA as a standard: it's just not as effective.
It is better to spike the RNA with a known, alien standard that does not occur in that organism prior to the reverse transcription reaction. Better yet, create a truncated synthetic RNA that is amplified by the primers you are using to measure the gene of interest so that you retain the primer reaction efficiencies. This isn't very practical for most people, but it is ideal. You can buy alien standards for real-time PCR from companies like Roche.
Using a consituitively active house-keeping gene, like B-actin or GAPDH, is also an effective method for relative quantitation.
I would see Wong and Medrano, Biotechniques 2005 (available online), for a good review on standards.
-Matt