QPCR - QPCR housekeeping gene (May/28/2005 )

sivasubi@hotmail.com

Hi
I am designing a QPCR expt. First I want to see if housekeeping genes are uniformely expressed in different tissues and treatments.
To check this may I use plasmid prep of different cDNA libraries instead of the tissue/treatments cDNA or RNA?
Siva

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Dear Siva,
I think using different tissues and treatments is more relevent to your study. However you still have to used you precloned plasmid as positive control in your QPCR assay.

Since you are measuring expression level of certain gene you have to do Rt-PCR.
Either one-step or two-step RT-PCR is doesn't metter. Most important thing is you have to standardised the amount of your starting material. For example, If you are doing tissue culture, do a simple cell count before you extract your RNA. Make sure that the amount of cells are around the same each time when you extrac you RNA.

regards
yong

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