Gene expression confusion - (Jul/28/2010 )

Hi!

Recently I was trying to measure the expression of a target gene (TGFBR1) using Applied Biosystem gene expression assays, with GAPDH as endogenous control. So after isolation of RNA with trizol reagent I did RT reaction and then set the gene expression assay. I didn't measure non of the cDNA nor RNA because I was thinking that when I use the endogenous control the normalization of the amount of RNA is going to be done according to the amount of GAPDH.
The problem is that one of the people working with me told me that sometimes if you dilute your cDNA, your gene expression assay may go much better. I did a quick research and I found out that it might be true due to the dilution of MgCl2 that is added to RT reaction and doesn't help the gene expression assay. By the way I thought that dilution would not affect the result of my expression assay because the ∆Ct will remain the same as both of the target gene and the GAPDH will be diluted. But surprisingly I gained totally different data for the same samples when diluted and not with differences of ∆Ct of more than 5 for example because the GAPDH was doing much better in dilution(earlier Ct) while target gene was doing worse , totally changing my results.

Does anybody have an idea how this can be explained? And which result is the correct one.

And help will be very appreciated.

Hmmm....repeat the PCR with the diluted and undiluted? Real-time can be finnicky, as it's pretty sensitive. With very dilute cDNA or very low expressing genes, real-time results start to get inconsistent (eg. Ct>35). The presence of MgCl2 in the cDNA shouldn't negatively affect the real-time PCR, as the amount would be really small, and MgCl2 is part of PCR buffer anyways and having slightly more or less shouldn't make much of a difference. Do you run melt curves and/or run the samples on a gel afterwards? We always do a melt curve after every real-time, and at least until the particular primers, cell type, treatment, etc. are optimized for real-time we always run a gel to make sure we're amplifying one product and that it's the correct size.

When doing regular PCR on gDNA isolated using 'dirty' methods (eg. no column') we've found that diluting the DNA can help the PCR, since it dilutes out contaminants that inhibit PCR, like phenol, so in some cases diluting the DNA can help, but probably not in this situation.

You should check your RNA using a spectrophotometer (eg. NanoDrop) before doing the RT, to determine concentration and 260/280 (and 260/230) ratios, and should make your cDNA using the same amount of RNA for each samples. Yes you will use Gapdh to normalize your samples, but ideally (and for more accurate results), this normalization should be minimal (eg. Gapdh between all samples should be within 1 Ct of each other, which shouldn't be a problem if you make all your cDNA with the same amount of starting RNA).

Check the efficiencies of your two different expression assays (TGFBR1 and GAPDH) by running a series of dilutions (undiluted cDNA, 1:10,1:100 ...) for both assays and creating a standard curve from it (efficiency can be calculated from the slope). Maybe the efficiencies are different, with GAPDH having a higher efficiency, although the two assays are commercial.

Hi, thank you for your posts so far.
My probes are FAM based, so I don't do a melt curve.
As for the efficiency it is supposed to be 1, as is stated by the company.

myself on Wed Jul 28 20:24:54 2010 said:

It´s better to do a standard curve when establishing a new assay. By doing a standard curve, you could check for differences in efficiency (although it´s supposed to be 100% but you never know), the presence of inhibitors in your samples and the linear range of your assay. Maybe the expression of your gene of interest is too low and you are out of the linear range. What CT-values do you get? Is somebody else in your lab doing the same expression assay?

I am the only one who usses it:/ I get Ct values between 25-37. I think I will just do the standard curve as you suggest.