RTPCR - housekeeping genes: can I use them more intelligently? - (Sep/14/2011 )
Dear all,
I am having problems (isn't everyone?) with my RTPCR experiments.
Method, briefly:
- I am taking tissue from mouse embryos. This tissue consists of derivatives of all three germ layers (endo-, meso- and ecto-derm).
- My genes of interest are all expressed only in the mesodermal fraction.
- Due to the preservation of the embryos in RNAlater the tissue is very fragile to the point where I have to 'dissect' my region of interest using pulled glass capillaries to tear the appropriate area away from the rest of the embryo.
- RNA extraction in TRIzol. Good quality on spec. and on a gel - no degradation and clean A260/280 ratios.
- cDNA produced using random primers from ~200ng RNA.
- Quantifying with SYBRgreen qPCR. Nice, tight replicates (3x for each gene).
- Using Pffafl method to estimate concentrations of mRNA compared to GAPDH.
Problems:
1. Very variable (spanning up to 2 orders of magnitude) values for the expression levels of the same genes when looking at different embryos from the same litter.
2. The crude dissection technique means that I get variable amounts of non-mesodermal tissue accompanying the mesodermal tissue.
3. As GAPDH is expressed in all tissue derivatives this means that samples which happen to contain more non-mesodermal tissue will have inflated GAPDH values compared to the genes of interest. This results in, I believe, the large variability seen across samples.
Solutions:
The best solution I can think of is to use something different for the comparison gene (GAPDH). I have several other housekeeping genes available (beta-actin, beta-2-microglobulin) but I was thinking - could I use something mesoderm specific as my comparison? This would effectively cut out the variability due to dissection and (hopefully) make my data far more interpretable. What gene to use would be tricky to decide, but is the idea valid in theory? Has anyone either done this or read about it being done? A cursory PubMed didn't turn up anything useful...
An alternative would be to FACS the cells prior to extracting RNA (if I can get some fresh tissue, not RNAlater preserved) by selecting for a mesodermal marker but I've never performed FACS before and don't even know whether the cells would be suitable for RTPCR afterwards.
Anyway, thanks very much,
J
Hi JWC,
If you are not familiar with the MIQE RT-PCR guidelines you might want to have a look as the section on normalization basically answers your question: http://www.rdml.org/clinchem.2008.112797v1.pdf
In short, using just one reference gene is dicey at best anyway, but more importantly is using a reference gene that is validated as stable in whatever sample you are using.
I did a quick Google Scholar search and this paper sounds promising:
Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells
doi: 10.1387/ijdb.052130ew
Good luck.
tl;dr yes
Cheers Delta, that looks like it will be a lot of help!
J
J_W_C: I don't see a DNase treatment in your protocol, do you have intron spanning primers tested for DNA amplification?
If you are looking for reference genes for a particular tissue type, the best is to use the free online tool RefGenes. It screens a very large database of high quality microarrays to find those genes that are the most stable in chosen tissue types, for example in mouse embryo. This is far better than testing a handful of housekeeping genes from a commercial panel. See also Hruz et al., 2011. RefGenes is freely accessible at www.refgenes.org.