RT-PCR of apoptosis related genes - (Sep/08/2006 )
I am trying to amplify fas, FasL, flip, FADD, Bcl2 , Caspase8 and Mcl from MCF-7 and DLD-1 cell lines after they've been treated with my molecule of interest. I know that at a particular concentration, both these cell lines have almost 100% death after 48 hrs of treatment. I can amplify Beta Actin from the cDNA but not any of the afore mentioned genes. My primer sequences are from published literature.
Does any one have any ideas, on what I may be doing wrong. As positive control for apoptosis, Ive tried treating the cells with etoposide (for MCF- cells, with 68, 102 and 136 uM for 24 hrs- ) and (DLD-1 with 50, 75 and 100 uM for 24 hrs). I've tried making RNA after washing the cells with PBS and in another attempt, after spining down the supernatant, washing the cells , trypsinizing them and then treating them with trizol. Since the Beta actin amplification works, I am assuming the RNA prep and the RT step are fine , and I may be doing something wrong at the treatment or harvesting step . For the life of me though, I cant figure out what.
Been reading up, and while one paper mentions fas being expressed cyclically in colon carcinoma, another mentions, FasL being constitutively expressed in colon carcinomas.
Is there any online resource besides the UCSC browser, where I can look up the expression data for genes.
I can provide the references I have if any one needs them . I have written to the authors of a few of these papers and am awaiting replies. Meanwhile, I'd be much obliged, If I could get some troubleshooting help here.
To answer your question about expression databases, you can try looking at GEO on the NCBI website, which will list relative amounts of gene expression within various tissues.
Since your actin control amplifies as would be expected I believe the RNA should be of sufficient quality for RT-PCR - so there must be some other issue with the primers you are using or with the expression levels of each mRNA. First thing is to check and double check that the primer sequences you are using are homologous to your transcripts of interest. I've had mixed luck using primers from the lit. Secondly, increase the number of cycles relative to the actin reaction. Actin is abundant, but the other targets may need 30+ cycles to visualize on a gel. This will take some optimization, but it could easily be why they aren't showing up for you.
I can say from experience doing RT-PCR for FasL that the timecourse must be carefully optimized such that you see transcript before massive cell death. The expression dynamics are a bit strange I think. You will also likely have to use MANY cycles as the mRNA is only expressed at very low levels, even in activated Jurkat T cells. I think my colleague uses just under 40 PCR cycles for her experiments.
Do you think your compound may be causing apoptosis via FasL induction? It may be easier to try and block it with a recombinant protein called FasFc, which is the extracellular domain of Fas fused to the IgG Fc region. Sigma sells it and several other companies. There are several antibodies that block Fas/FasL interactions as well.
Just so you know, many cancer cell lines are actually resistant to Fas mediated apoptosis (its a selective advantage)
Hope this helps,