cDNA and RT-PCR - (Feb/03/2009 )

I am having trouble with real time PCR. I tried just the beta-actin on my cDNA to see if I could get that to work and I get no amplification. The quantity and quality of the cDNA looked fine according to the Nanodrop. The primers are common for our lab and nobody else has trouble with them. I noticed the cDNA has to be mixed up before use because I see precipitate at the bottom of the tube. Is this normal or could this be the problem? What should I do to make them more soluble, do they need to be warmed up? Any ideas are appreciated.

Measuing cDNA on Nanodrop won't tell you anything. You should first check your RNA quality by running a agarose or denatured agarose gel to see if your RNA is degraded. If RNA is fine, amplify some other cDNA sample know to be good to see if your PCR system is fine. If yes, then the problem is at your RT reaction.

Just to clarify, are you running real-time PCR, or reverse transcription real-time PCR? Am I understanding correctly that you are using cDNA as your starting template?

Stedda on Feb 4 2009, 10:22 AM said:

So the concentration and 260/280 ratio isn't enough to tell you if it's OK? I'll try running a gel. What about the precipitation when I defrost it? Is that normal? Thanks.

pcrman on Feb 3 2009, 09:44 PM said:

It is real time PCR and cDNA is the starting template. Thanks.

Unagi on Feb 3 2009, 11:18 PM said:

Stedda on Feb 4 2009, 01:22 AM said:

cDNA, especially when fresh-amplified, shouldn't precipitate like that, being solubilized in buffer/water.
Are you sure everything's ok at this point? Though...what could be wrong.... (do you heat up water with cDNA in it or leave it in the fridge in 4 degrees C. overnight?)
And I agree you should try to run a gel and check RNA quality first.

I don't heat the cDNA up and I store it at -20. Though, I can't remember if I thawed it on ice or not...that may be the problem if I forgot to do that.

unity on Feb 4 2009, 12:38 PM said:

Stedda on Feb 3 2009, 05:22 PM said:

cDNA should not have precipitate, you are quite right, it is the trouble part. I don' think you should waste time in solublizing it. Get new aliquot or make the whole thing from scratch----unless a reviewer is waiting on your shoulders!!

Freeze/thawing too many times can start to degrade the cDNA, but as far as general thawing, I don't think doing it on ice is necessary.

Not knowing the entire scenario, I can suggest a few options:

1) as previously posted, try again with a freshly amplified cDNA library, if possible
2) if not, try a simple serial dilution series of the template, and run the beta actin assay with them - could be an issue of an introduced inhibitor
3) If you are really stuck, then an ethanol precipitation of the warmed up cDNA may help.

Stedda on Feb 5 2009, 10:15 AM said: