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18S changes with cDNA dilution


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#1 cmi

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Posted 08 June 2009 - 07:44 PM

Hi - help!
I am using 18S and beta actin as HKG across three treatments. What I have found is that the Cts are constant across these treatment groups, indicating these genes are appropriate HKG when I dilute the cDNA at 1:50. HOWEVER when cDNA is diluted 1:75 there is a significant difference in the expression of both 18S and beta actin across treatments, but not within treatments. This is a dilemma, as it suggests that these genes are not appropriate HKG. Does anyone have an explanation? or a recommendation?

#2 pcrman

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Posted 08 June 2009 - 11:10 PM

I don't think there is a real difference in HKG expression between treatment after dilution. It is likely after dilution, the PCR system is more sensitive to template differences. We can put it this way: before dilution, you have unequal RNA loading and your PCR did not pick up the difference between treatments in housekeeping genes. After dilution, your PCR is now able to tell there is a unequal RNA loading issue.

#3 cmi

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Posted 09 June 2009 - 01:01 PM

I don't think there is a real difference in HKG expression between treatment after dilution. It is likely after dilution, the PCR system is more sensitive to template differences. We can put it this way: before dilution, you have unequal RNA loading and your PCR did not pick up the difference between treatments in housekeeping genes. After dilution, your PCR is now able to tell there is a unequal RNA loading issue.



Hi pcrman,
Thanks, I thought that might have been the case. I will now screen various HKG - but of course may not come up with one that is appropriate. I'm tempted to just publish delta Ct relative to input RNA but given the feedback I get when I suggest this in discussions with various people, I expect quite a bit of resistance from reviewers/editors. Have you had any experience with publishing relative to input RNA?




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