Hey there, I wonder if anyone can help me with the following issue: I’ve been running recently qPCR reactions on cDNA prepared from intestinal epithelium-enriched samples and ran into a recurring problem that I’ll explain using an example from one of my experiments: in that experiment, I’ve had samples from 3 KO and 3 WT mice, and in every qPCR reaction I’m running (on cDNA samples reverse-transcribed on several different occasions) one of the WT mice, which I have randomly called “WT3” keeps getting much higher Ct values than all 5 other mice for each of 3 different housekeeping genes I’ve been trying (Hprt, B2m and Ubc; and also for mouse target genes). For instance, the Ct values for Hprt in one qPCR run were ~21.8-~22.8 for all mice except for WT3, for which it was ~26.8. The increased Ct for the housekeeping gene in this mouse results in normalized expression levels of most target genes in this mouse which are dramatically different than in the other WT (and KO) mice.

A few extra details: the RNA was extracted using TRIzol and quantified via Nanodrop. Mouse WT3 yielded a much lower RNA concentration (~225 ng/uL) than all other mice, but it was still sufficient for many qPCR reactions. Equal RNA quantities were used for the RT-PCR reaction (1 ug RNA for each mouse, using the “High-Capacity cDNA Reverse Transcription Kit” from “Applied Biosystems”), and the resultant concentrations of cDNA (quantified by Nanodrop) were highly comparable between all samples/mice. For RNA, the 260/280 ratio obtained for WT3 (1.83) was somewhat lower than for all other mice (1.95-1.99), although the 260/280 ratio of the cDNA was almost identical between all mice, including WT3 (1.83-1.84).

Anyway, three questions:

1. What could be the reason for the much higher Ct values in one sample compared to all the rest?

2. What, if anything, can be done to rescue this sample?

3. What, if anything, can be done to avoid similar situations in the future (with new samples)?