Of course i need two step RT PCR

Hi,

On the first step you do the RT: your RNA is mixed with the RT-buffer and Reverse Transcriptase, as well as with Random Primers/oligo(dT) Primers (depends if you want to get whole-transcriptom cDNA or only cDNA from poliA-containing RNA (mRNA)). Usually it is 45-60 mins at 42 degrees with heat-inactivation of enzyme at 72 degrees.

But in general you can simply follow the RT-kit manual.

For ultra-low RNA concentrations (hundreds/thousands of FACS-sorted cells) I can suggest PrimeScript from Clontech (RR037A), for more concentrated samples you may use various of other kits, I used SuperScript II from Invitrogen.

You may also clean-up your cDNA with a column (for ex. Qiagen MinElute) or magnetic beads (for ex. AmPure XP).

The next step is to do the PCR itself. If you go for gene-specific PCR, you just need to add PCR MasterMix (for example, SybrGreen 2x if you use the qPCR machine) to your cDNA aliquot as well as Fw&Rv primers, covering the fragment of the cDNA of the transcript of interest (but which are not working with the DNA itself - be careful during Primer design and use DNAse treatment during RNA isolation, if you use columns). Run 20-40 cycles, depending on the expression of the target gene. Don't forget to make technical/biological replicates, as well as to run the controls (GAPDH/etc.)

Best,

Michail

My previous lab used iScript (Biorad) for the cDNA synthesis. It follows a similar procedure to this: http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3911

The rest are just regular PCR steps.

Dear Zahra

you can follow this paper:

Bachman J. Reverse-transcription PCR (rt-PCR). InMethods in enzymology 2013 Jan 1 (Vol. 530, pp. 67-74). Academic Press.

Best

Hi, please see the following link

ftp://ftp.sanger.ac.uk/pub/resources/mouse/sigtr/RTPCR.pdf

Thank you.