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18S as a housekeeping gene for RT-PCR? getting wide variation


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#1 medstudent1

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Posted 03 April 2009 - 01:02 PM

hello,

i am doing a few RT-PCR experiments and i am having some trouble with my housekeeping gene (18S). my ct values for my unknowns over the course of thee experiments are fairly consistent however my 18S is varying quite a bit. im not sure if 18S is known to be quite variable.

it is making the data quite difficult to interpret as my error bars are huge. if i had tight Ct values for 18S, things would look much better.

not sure if i am doing anything wrong since my sample Cts are all consistent and the only thing that is not is my 18S Ct values.

any suggestions?

#2 pcrman

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Posted 03 April 2009 - 08:50 PM

Have you run regular PCR using the primers for 18S rRNA or run the real-time PCR product to see if it gives you an unique band. You should also run a disassociation assay to see how the primers behave. 18s is supposed to be a better control than beta actin.

#3 Ahrenhase

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Posted 03 April 2009 - 09:14 PM

18S should be fairly constant. I did a test on samples treated with a harsh toxin and 18S more consistent than GAPDH and b-Actin. I guess it all depends what the unknowns are treated with.

have you ran a melt curve following the reactions to see if you get one peak?

Also have you optimized the 18S primers to make sure they are properly amplifying throughout each step.




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