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What is the ideal amount of RNA to use for the RT? and how much cDNA to use then for the PCR?

I did RT with a solution of RNA of 0.36 ug/ul. Then for my PCR I used 1 ul of the cDNA obtained and used 25 cycles because I was hoping to see a difference between an endogenous level and an overexpression. However, I detected no band at the expected height of 500 bp but a diffuse band at 100 bp. What can this 100 bp represent? primer dimer? I switched to 35 cycles and I used either the same amount of cDNA or add 3 times more cDNA. In both cases I got bands at the expected height (but stronger when I put more cDNA) and still big bands at 100 bp.

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RT PCR refers to reverse transcription I assume? Not to real time PCR? –  Gergana Vandova Jan 12 '12 at 8:50
    
yes! I should have be more accurate. Thanks –  kali281 Jan 12 '12 at 8:58
    
@Alexander Galkin Why did you remove the PCR tag? I think the question is really about RT PCR, and not RT in particular. –  Gergana Vandova Jan 12 '12 at 19:35
    
@GerganaVandova Do you think that we need a special tag for RT PCR? Isn't it too confined area? I wasn't sure whether to correct it "PCR" or to "reverse transcription" and chose the last one. If you feel it is wrong, feel free to retag the question, it is more than welcome in the community. –  Alexander Galkin Jan 12 '12 at 19:40
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@Alexander Galkin Yes, I believe the question is more about PCR rather than RT. You are probably right that RT-PCR tag is too specific. The PCR tag is good enough. –  Gergana Vandova Jan 12 '12 at 20:04
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2 Answers

up vote 6 down vote accepted

I see big fuzzy bands around 100bp as well. They're most likely RNA contamination. To get rid of them, digest your RT-PCR products with RNAse-H. But if you just need to visualize your band of interest, and the fuzzy bands aren't getting in the way, it shouldn't be a problem.

I usually input anywhere from 1-2 ug of RNA into my RT-PCR reaction using the Invitrogen Superscript III kit for a total volume of 20 ul. After the reaction, I use 1/10 the volume of that (2 ul) for downstream (PCR) applications. This usually gives me nice results.

To optimize RT-PCR for detection of a specific target, consider using gene specific primers (make sure to use only anti-sense primers) in your RT-PCR instead of oligo-dT or random hexamers. This enriches your target when you use them for downstream applications. When you use GSPs though, make sure to run a parallel reaction with control (i.e. beta actin) primers to prove that your RT-PCR reaction is working.

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There is no single "ideal amount" of RNA. I would suggest you to do a titration curve to determine the best amount for your specific assay. The band at 100 bp could be a non-specific amplification. You could try to increase slightly the annealing temp to see if that removes (or reduces) the lower band. Alternatively, you can consider a different primer set or even a nested PCR. Also, it is still possible that you may look at a splicing alternative in that band. Do your primers span more than one exon?

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