3 replies to this topic

[thenovice]

Hi I have two questions:
1) I ran real time RT-PCR from a year old RNA sample stored at -80⁰C and failed to get any signal for the genes of interest ASIC and TRPV1 (Pain sensing genes). that is when I pressed the "analyse" button, these genes were "undetermined" while the house keeping gene (GAPDH) showed Ct values of 21-23.
can anyone tell me what the reasons could be?
2) I have a cDNA sample of the same RNA mentioned in question number 1, and  was stored at -80⁰C for over a year and left unused. Is it wise to use it for real time PCR? Is it stable at -80⁰C for such a long period of time. And can I expect to get any signal for ASICs and TRPV1 if I use this cDNA for real time PCR
FYI: When we ran PCR (not real time) from the cDNA a year ago, we had got good bands with the PCR products
Hoping to get some answers
Thanks
Maria

[kajmak]

I have never done RT-PCR but guy in my lab always uses cDNA as a template and I am pretty sure he stores his at -20, probably only because it is easier to open that freezer.  
But he does it in batches. Number of RNA extractions, cDNA, RT-PCR and then the next round.
Nothing should happen to RNA at -80, but after some time it gets messy.
Maybe there was not enough RNA of your genes, compared to housekeeping gene.
But, if my colleague is doing cDNA step there must be a good reason for that.

[scolix]

I will go ahead with the RTPCR with cDNA. Normally I make cDNA and use it for further analysis.

[thenovice]

Thanks all... Thats a lot of help
Thanks