I would like to know if the reverse transcription reaction influence or not the DNA expression, in other way I would say which appropriate protocol to convert RNA to DNAc for real time PCR reaction, because i found many protocol with different time of incubation, could you suggest good and not expensive one, Regards.
Hi, I worked with this: http://www.bio-rad.com/de-de/category/pcr-reagents/reverse-transcription-reagents?WT.srch=1&WT.mc_id=aw-gxd-eu-pcr_iscript-reverse-transcription&gclid=CLSc16LA18ECFagKwwodlIoALQ&WT.knsh_id=868482f9-2d2d-4043-ad9e-ba5a0f58877c
It worked very well. Cheers, Nadine
Thanks Holger, but iwant to just make some clarification, in my PCR i would like to know just the expression of some specific genes (using my own designed specific primers), any suggession please, regards.
I use ToBoyo kit for cDNA working fine for us and we use our specific primers
and then Sybergreen QPK201
This booklet from Qiagen has a very good introduction to the various techniques used to perform quantitative real time PCR. http://www.scribd.com/doc/59157839/Critical-Factors-for-Successful-Real-Time-PCR I've had good success with the NEB PrimeScript II kit as well as the Invitrogen SuperScriptIII kit for making cDNA. I've used SYBR-Green to do the qRT-PCR on an ABI machine. You might also like to be aware of the MIQE Guidelines http://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf
Hello Younes,
If you want to statrt with specific primers for your RTase experiment, if possible pick up primers from 2 different exons framing an intron like this you will make the difference between RNA and DNA source of your amplification and it will let you know about DNA contamination or not.
If not you start with branched oligodT that 3' terminates by G,C, or A but not T like this the amplification will start at the junction of the polyA motif and the 3'UTR sequence.
For the kit both previous advices work well.
Good luck !!!
Edouard
.
Incubation time is critical for cDNA conversion efficiency. And efficiency has a huge effect on downstream applications like qPCR expression assays. Really I can't stress this enough. qPCR is a pain in the butt for this very reason.
Longer incubation times are always better. When in doubt tack on more time to the incubation.
I personally use this kit: http://www.lifetechnologies.com/order/catalog/product/4368813?ICID=cvc-rt-rt-pcr-c1b1
Their protocol suggests up to 2hrs incubation time at 37c for up to 2ug RNA. It has worked flawlessly every time. Biggest thing is to have a good idea how much RNA is going into each reaction. And make that amount consistent from sample to sample. So I personally use QUBIT for RNA quantification, because it's more accurate than nanodrop. Also if you have a bioanalyzer available, you should probably QC your RNA to make sure there aren't integrity issue (as this can also have a huge impact on qPCR performance, but not necessarily cDNA synthesis)
There are a ton of factors which influence qPCR performance, so please use me as a sounding board if you have any more questions, troubleshooting issues, etc.
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