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Multiple internal controls in using qPCR to measure gene duplication

gene duplication gene amplification qPCR internal controls

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#1 hongkongrubbish



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Posted 03 November 2011 - 06:51 AM

Dear all,

I am having a project to investigate a gene amplification/duplication in Hepatocellular Carcinoma (Liver cancer), and I am planning to use Real Time PCR to measure the genomic DNA content of that gene in samples.

However, the increase in the number of gene copy (DNA) will be much smaller (can be as few as 1~2-fold) than that in overexpression of mRNA (up to several thousand folds). So my boss asked me to get more than one internal control (or reference) genes and take geometric average to ensure accurate measurement.

Then I have spent several weeks searching literatures, some studies used a few genes in RNA (or cDNA) measurement, but none of them reported to have used multiple internal controls in measure DNA (they all used one only). I gave my boss the list of genes that people used in RNA, but she didn't accept because she said those worked on mRNA measurement did not mean they would work on DNA. She wanted some published ones and thought that was easy....

I am desperate now... Does anyone have any ideas on what I should do?? Thanks!!!!!!Posted Image

#2 merlav



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Posted 08 November 2011 - 11:33 AM

Usually when doing QPCR of DNA the IC or reference is a Housekeeping gene that is very like to be present for example: ß-actin, GAPDH, others use also TUBA3. I use ß-actin label with a different dye than the gene of study so I can do a multiplex.
Science without religion is lame, religion without science is blind.
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I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

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