I've done isolation of total RNA from several testis samples, reverse transcription and in the qPCR. I got no signal from the housekeeping gene (GAPDH) in 30% of my samples; however, I got the signals from the gene of interest (Ct ~20). After the 10-fold dilution of cDNA, I got the GAPDH signal - that means inhibition. The mRNA preparation is a combination of Trizol and RNeasy columns procedure.
Where do you think the inhibition comes from?
It is quite surprising!? Is there correlation with particular treatment of mice?
I have isolated total RNA from various testis samples (fetal and adult rat and mouse, fetal human), and I never had any problems. I use RNeasy columns for isolation and 18-S is my housekeeping control gene in my real-time qRT-PCR experiments.
The most probable source of inhibition in your case is the cDNA itself.
Diluting the cDNA little or not at all causes inhibition, that is not necessarily equal for all targets, as it happened in your case.
A very simple and straightforward procedure you can do in advance, before subjecting all your cDNA samples to qPCR, is to make a pool of equal parts of cDNA from all samples and then make serial dilution of that cDNA pool and run several genes of interest for each dilution point.
In this manner you will get a quick and preliminary view what is the linear quantification range for each individual GOI and then you easily decide what is the dilution that is "good" for all GOIs.
After doing this several times with different tissues, I now almost always dilute my cDNA in a way that I have 1ng cDNA (RNA equivalent) in each qPCR reaction well.
I guess this depends on the combination of reagents everyone is using.
Thank you for your answers. The inhibition was not correlated to the tretatement of animals.
Petar, you hit the bullseye. I've already conducted several test in a way, you propose it. Unfortunately some of my GOIs have very high Ct, thus the cDNA dilution to meet the linear range of my reference gene resulted in undetectable signal of one of the GOIs
The cause of inhibition was to high amount of RNA added to RT. The new RT with lowered RNA concentration helped. After that the linear range begins from undiluted point of the standard curve.
Hi Maciej, I'm glad that you already resolved the issue!
Reducing the RNA in the RT was the plan "B" :) But indeed, I am more and more strongly convinced that "Less is better" in qPCR.
In fact, the same dilution series I suggested can be done with the RNA - retrotranscribe various quantities of RNA and do qPCR with a well-established assay for a GOI. In this way one can actually determine the linearity of the RT step.
Of course the outcome of this RT linearity test will depend on the matrix (tissue) and the RT enzyme used, but I believe it worth the effort, because it saves a lot of time in the long term...
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