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Ct fluctuations in qPCR


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#1 ela0405

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Posted 15 May 2009 - 12:26 PM

Hi,
I wonder if someone can help me.
I`m trying to validate a microarray on drosophila genes. My problem: Over half of my genes show no conatant Ct values. That means that 3 independent extracted RNA samples of the same genotype show (although I normalize) ct valuations over 3 Cts.
So I cannot evaluate the data.
I've already done troubleshooting and I do not know how to continue.

I extract the RNa from larvae wit the RNeasylipid extraction Kit from qiagen. I make an on column DNase I digestions. The resulting RNA is really good. Another person has also done it (no faults from my way). Then i put about 1 g RNa in a CDNA synthesis with Iscript (Biorad). After purification with Nucleo Spin eXtract II (macherey and nagel) the CDNa is put in the Real time 1:20 diluted.
I`ve checked the extraction, changed all kits, changed the DNaseI, looked at the dissociation cirve, morphological selected the larvae.
I`m wondering if they are any problems due to the fact that the genes I try to validate are really small (down to 200bp).

Has anyone an idea? The CTs of my refernece gene does also fluctuate.

Thank you for helping me!

#2 asdfyrr

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Posted 17 May 2009 - 06:45 AM

mybe your pcr /primer efficiency is not very good. do u make a dillution row of the dna samples to check the efficiency during each run?

#3 ela0405

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Posted 17 May 2009 - 10:16 PM

Thank you for answering....
I checked my primers in the beginning with 5 different dilutions of CDNA....they looked really good. Additionally the dissociationcurve shows no primer dimer or something else...ave you ever heard of problems within the quantification of small genes?

#4 asdfyrr

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Posted 18 May 2009 - 11:43 PM

Thank you for answering....
I checked my primers in the beginning with 5 different dilutions of CDNA....they looked really good. Additionally the dissociationcurve shows no primer dimer or something else...ave you ever heard of problems within the quantification of small genes?


no sry i have not.
when i do RT-qPCR, i always do a dilution row for each primerset (i.e for each experiment). thus i can determine the reaction efficiency, and only reactions with comparable efficiencies have also comparable Ct values.
the same reaction mix would give different ct values if the efficiency is in one reaction 100% and in the other 70%, even if it would start with the same number of templates

but if the efficiency is always around 100% and comparable between reactions...then I gave no further ideas, Im sorry




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