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cDNA and RT-PCR


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#1 Stedda

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Posted 03 February 2009 - 03:22 PM

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.

#2 pcrman

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Posted 03 February 2009 - 08:44 PM

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.

#3 Unagi

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Posted 03 February 2009 - 10:18 PM

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?

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.



#4 Stedda

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Posted 04 February 2009 - 09:55 AM

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.

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.



#5 Stedda

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Posted 04 February 2009 - 09:56 AM

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

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?

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.



#6 unity

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Posted 04 February 2009 - 11:38 AM

I noticed the cDNA has to be mixed up before use because I see precipitate at the bottom of the tube.

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.... B) (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.

Edited by unity, 04 February 2009 - 11:39 AM.


#7 Stedda

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Posted 04 February 2009 - 03:15 PM

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.

I noticed the cDNA has to be mixed up before use because I see precipitate at the bottom of the tube.

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.



#8 TanyHark

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Posted 04 February 2009 - 07:51 PM

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.

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!! :(

#9 Unagi

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Posted 06 February 2009 - 03:24 PM

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.



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.

I noticed the cDNA has to be mixed up before use because I see precipitate at the bottom of the tube.

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.... :D (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.






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