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qPCR and varying cDNA concentrations


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#1 starvingstudent

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Posted 17 August 2009 - 09:12 AM

Hello,
I ran two different qPCR rxns, the protocol I'm using calls for a 1/10 dilution of my cDNA of which 5 uL goes into the rxn. The first time I ran the rxn, I found that I have 500,000 transcripts in a given tissue (I did two replicates and they were both pretty much consistent around this value). I ran a second reaction again with the same cDNA, this time I found that the tissue has 1,000,000 transcripts. I suspect this discrepancy may be a result of an error when diluting the cDNA the second time. Perhaps, made a mistake and added more cDNA then I should have?

I just want to know if anyone has encountered this sort of problem and if it's anything else besides how much cDNA i used?

Thanks!

#2 cellcounter

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Posted 18 August 2009 - 08:39 AM

Hello,
I ran two different qPCR rxns, the protocol I'm using calls for a 1/10 dilution of my cDNA of which 5 uL goes into the rxn. The first time I ran the rxn, I found that I have 500,000 transcripts in a given tissue (I did two replicates and they were both pretty much consistent around this value). I ran a second reaction again with the same cDNA, this time I found that the tissue has 1,000,000 transcripts. I suspect this discrepancy may be a result of an error when diluting the cDNA the second time. Perhaps, made a mistake and added more cDNA then I should have?

I just want to know if anyone has encountered this sort of problem and if it's anything else besides how much cDNA i used?

Thanks!

There are so many reasons this could happen, for example:

(1) Pipetting error

(2) Change in the reagents (fresh ones).

(3) technical variation (i.e. changing the total volume of reaction).

(4) but the most likely is, water in the tube containing cDNA had condensed and you didn't spin it down before using it, or the cDNA had dried up a little bit, increasing the total amount of transcripts/ul. This may be taken care of if you normalize against housekeeping gene, but depends upon how you calculate the number of transcripts and compare with the previous experiment.




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