About housekeeping gene - (May/09/2007 )

hi, i am doing a project in screening with secretion of Heat shock protein 70s mRNA at different condition with fungus....i got a problem recently. No bands were observed in gel after running PCR. We figured out two suspected factors. The cDNA synetheized in Reverse Transcription or the degenerate primer used in second-strand synthesis.
My supervisor told us using housekeeping primer to check the cDNA. i want to know the principal of house keeping primer and the reason to use it to check cDNA...

can anyone answer my question???
thx~~

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Hi larrychung,

A housekeeping gene should be expressed under all conditions e.g. GAPDH, B-actin etc (there is some controversy about their universal expression under certain circumstances like e.g. hypoxia but in general they are ubiquitously expressed). Therefore if you get a PCR product for your housekeeping gene you know that the first strand cDNA has been correctly synthesised. Housekeeping genes are also used to check for equal loading etc.

Hope this helps,

Auldmok

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i use beta-actin gene primer for fungus. if about 1.7 kb fragment by amplify then i can understand my reaction is ok.
actin express equally under all condition so you can compare your sample or culture condition etc etc

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Thx both of you to reply my question~~

As i know the first-strand cDNA which is not the cause of failure of second-strand synthesis.
i can conclude that the problem is due to the degenerate primer, but i wanna know the reason that degenerate primer to cause this problem.
can anyone explain it??
thx~~

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