3 replies to this topic

[wrch]

Hi, I have just done a real time PCR with 18S as reference gene. while the Ct value of my target gene is between 22 and 23 the Ct value of reference gene is just about 5. If I reduce my template then the Ct value of the target gene will be higher also accordingly. Is it acceptable to use such low Ct value for reference?? If not what can I do other than to change the reference gene??
Please help.

[Trof]

No it's not acceptable. Acceptable range for quantification is around 15-30 cycles.
Decrease concentration of your 18S primers.

[wrch]

will dilution of primer only serve the purpose?? Is it possible to use different amounts of templates for target and reference gene?

Edited by wrch, 03 April 2012 - 07:09 PM.

[Trof]

It should, to some extent. If you use different amounts for reference and for your gene, you're not normalizing it, because it's now a different sample. Because you would need to dilute it like 1/1000 for 18S.

Dilute your cDNA 10 times for for both genes, this would shift Ct's for about 3 cycles, still acceptable for your target gene, and then try to limit 18S primers. If it's not enough you would need to choose different reference, the problems with 18S in this regard are well known.

Other thing to try could be using oligo dT primers together with random hexamers for RT, and using less hexamers, but I'm just wild speculating.