Is it necessary to quantify RNA every time before cDNA conversion or can we just quantify cDNA after reverse transcription? How much difference does it make in relative quantification experiments?
Basing on my experience with tissue RNA isolation I would strongly recommend to quantify the isolated RNA. To high RNA concentration can cause problems in RT and later in qPCR (inhibition). The good idea is to follow the reverse transcriptase manufacturer specification.
Hi all, Is it true that RT enzyme can bias its reactivity towards some mRNA transcripts than other transcripts?
You should not only test your RNA quantity, you should also test your RNA quality!!!
RNA Integrity Number (RIN)!
Cheers, Nadine
If you have high quality enzymes and/or kits, then you have the chance that RT works fine, like 100% of the DNA is reverse transcribed or so. So, yes, it is good to have the same amount of RNA, which, yes, necessitates the quantitation of the RNA.
Moreover, in this case, if you start all parallel reactions with the SAME amount of RNA of the SAME quality, you will have the same amount of cDNA, with only the pipetting errors. The pipetting error is due to be something minor, and even if you pipette few microlitres of the various ingredients to make up the 20 ul reaction volume, the pipetting error should be less than a few dozen of percent. It means, that you have the chance to start all QPCR reactions with the same anount of cDNA with (let's say) 10% error.
Now, why it is extremely handy, is that even the best housekeeping genes can sometimes have significant (or even dramatical) changes in their expression levels , especially if you have some rude stress conditions involved in the study or something similar. Relying on (control) gene expression that may never been thoroughly investigated under your experimental setup is not safe. What people most often do is that they carefully investigate several such genes and choose a few (more than one) with the most stable expression to avoid the accidental problems stemming from altered expression of their favourite (one) control gene.
But if you have the same amount of cDNA as the derivative of the same amount of mRNA in all your reactions, this problem just can not occur (or at least you will not accidentally pass over it). You may still need to use an internal control gene for security and/or comparison, but at least there is a more or less safe way of making sure that your control gene expression has not changed significantly.
So, in brief,
1., make sure your RNA is of high quality and purity (and completely free of gDNA traces),
2., measure it's concentration, and
3., use the identical amount in all RT reaction, and then
4., use identical amount of cDNA in all qPCR reactions.
Thank you, Peter Kos, for ur elaborate and detailed advice... but my concern here is.... How should I purify mRNA from tRNA and rRNA in bacteria. As u know, there is no poly A tail at 3' end.... So, If we isolate whole RNA from bacteria, the concentration of RNA is cumulative of mRNA, tRNA & rRNA, isn't it? How much correct would be my results, when I use same concentration of whole RNA from treated and untreated bacterial culture? Bcs, the amount of tRNA and rRNA extracted may vary from sample to sample, doesn't it?
You are right, there is a chance theoretically that during the treatment or whatever experiment the ratio of mRNA:tRNA:rRNA changes. In our practice it has has never caused any problem though.
I can not rule out the possibility of that to happen. Still, only a very small fraction of RNA has regulatory function in itself, and the rest is used for protein synthesis. Therefore even if the growth (doubling time, overall protein synthesis, etc.) changes a lot, the same distribution of the above three types of RNA would be needed. This is only a speculation, though. What is certain is that in most of the cases the above ratio does not change, and you do not need to worry about that.
On the other hand if you want to get rid of rRNA, there are methods for that like substractive hybridization and such, but these are only necessary in whole transcriptome sequencing to make that cheaper, and not at all in gene expression studies.
You have already so many good answers that you don't need mine. I can only say that we always quantify RNAs before RT but also we check the quality that in my view is one of the most important steps. It is also true that it is important to use the best reagents you can as to have reproducible data. Our RT done both using biological triplicates and technical triplicates give always more or less the same results. Good luck
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