As far as I remember, superscript IV troubleshooting suggests prolonged (

• Thanks Asta, I did the SSIV reaction as suggested at 55 degrees and still nothing from PCR. I am very confident in my PCR because I have three positive controls of other genes from same RNA sample that work really well, I think because their transcript is much lower in GC content; I have sequenced one of my positive control extracts to ensure it's a real positive control and was 100% correct as predicted. And I know the gene I am trying to amplify is definitely more abundant than the other two genes I use for positive control. I also tried gene specific primer for that, and got a smeary band on my EtBr. I can see something very faint around the weight of my amplicon, but its definitely not pure and I don't think trying to cut around that gel will be useful especially since so much is lost in column purification.

Any MMLV enzyme can use for RT. You can use any hot start tax pol. for >%65 GC content. In addition to, you can increase the denaturation time for PCR. It works most probably.

So, because the controls are lower in GC content, you are getting the PCR fragment and it shows that the system works for that %GC.

Have you thought about PCR high-GC control?

I did some back-thought thinking :)

In your case, I'd make PCR control reaction with >65% GC DNA fragment.

Then, use random hexamers + 55°C + 1h  for cDNA synthesis. Then use varied amount of cDNA for PCR. Be wary, though, the hexamers might result in additional short PCR fragments.

Hope this helps. 

Yes, you can increase the initial heat denaturation step of the RNA (before you add the RT enzyme!), there may be a risk of some denaturation, but you would not need to heat for very long to destabilise RNA structures.

If you are asking if you can do the reverse transcription above the temp recommended by the manufacturer, then the answer is no. The reason for this is that the RT enzymes are not thermostable, ie they themselves will denature at higher temperatures and thus be non-functional. You may be able to increase the RT temp a few degrees but I would expect reduced RT efficiency in response.

If you are asking if you can increase the primer annealing temp in the PCR reaction, in principle yes, but not above the recommended extension temperature of the polymerase you are using and obviously dependent on the primer melting temp itself.