I have problem about the number of control samples in each of the steps of stem loop qRT.
In the 1st step (Stem-loop RT), 2 types of controls are required [no RT control and no RNA control]. I am working with 3 miRNAs and 2 different RNAs [tRNA]. So the total samples I should have are 6, without the controls. How many control treatments should I have in this step?
In the 2nd step [end point PCR], there is a 'no RT product control', so how many samples for this step?
The problem is that with this much number of samples, I can't run the qPCR [which can take the maximum 96 number of samples], while my samples might be more than 100 (according to the calculations). So what should I do?
Another question is about the 5S control that is usually used in the qPCR to analyse the results. Does that also accounts in the steps or not?
Hi Muhammad!
From my view, in the 1st step, the no-RNA control is unnecessary. Because, even if you have contamination with gDNA (even after the DNAseI step that is supposed you should have performed), the stem-loop would not be capable to amplify DNA. Also, at least in the protocol that I use, the low temperatures used to make cDNA from miRs using stem loops, avoid the possibility of breaking both strands of DNA, thus making a DNA-based unwanted amplification impossible, at least in principle (one does not think there are ssDNA around there!). It takes one control off at this stage.
I assume from your question that you have 5 genes of interest to amplify (3 miRs and 2 tRNAs). I don´t understand how you reach up to 100 samples, because you didn´t specify the number of treatments/gennotypes you want to use to compare miR/tRNA relative abundance.
But, in the 2nd step, I would use the RT control only once, for example for assessing the amplification of the Reference gene (5S, for example). You can perform the amplification (real-time, your 2nd step) of the 5S for all your experimental subjects (triplicate or, at least, duplicate) and include in that amplification (and only in that one) the RT- control. If good amplifications are achieved in all wells except in the RT- one, then you can conclude that it functioned well. It has no sense to check the RT- every time, in my opinion, as it is a waste of plate space, time and resources.
If you are entering the qPCR world, I strongly suggest you to read some good papers about reference gene selection (I ´ve personally found the 5S very complicated to amplify (in Arabidopsis at least) and I use EF-1a, but using Bestkeeper, GeNorm, Normfinder or other will help you to decide, check the bibliography!). Also, papers by Pfaffl (google it + ddCt, etc) are a good basis to understand and will save you a lot of time and money. Because if you perform flawed experiments, reviewers could ask you to do everything right form the start again, in 4 years from now!
Also search the internet for MIQE rules in qPCR experiments and read it all BEFORE you plan your experiments, Follow these rules at the great extent you can.
Sadly, lot of people give low importance to the details, but qPCR exeriments are delicate and the results could change completely if appropriate guidelines are not followed.
Best luck!
Thank you very much for a detailed answer Carlos. I performed the stem-loop RT with only 1 no-RNA control and full no-RT control. After I checked it with simple End-Point PCR, i realized that there was amplification in both normal and control samples (the no-RT control of the previous step). After asking some seniors, I realized that I missed a step about tRNA treatment with DNAse. Since then, I am waiting for samples to come because I used up all the tRNA i had..
I will follow your suggestions from now on, they are really precious. Thanks alot
What about designing the specific primers for stem-loop RT & qPCR? how to test the quality of primers using softwares?
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