Experiments with Small Cell Lung Cancer
Once we had decided on a disease model, namely Small Cell Lung Cancer, we needed an in-vivo way of testing our system. To this end we obtained the H82 Cell line from ATCC.
The first thing we had to ensure was that we were able to get DNA into the new cell line. We performed a Viafect optimization to test what our ratio of plasmid to transfection reagent should be. We transfected constitutively expressed BFP and reagent at ratios 1:1, 1:2, 1:3 and 1:4.
Ratio of Viafect:BFP vs. the blue fluorescent output (AU) of the H82s
We see the most fluorescent output at a 1:3 ratio of Viafect:BFP. Moving forward, we transfected at a 1:3 ratio.
To analyze the effectiveness of our RNA binding protein system on the alteration of the splicing event of REST in H82 Small Cell Lung Cancer cells, we conducted to Real-Time PCR on H82 SCLC cell line samples, transfected with the dCas113a along with the constructed REST1, REST2, REST3, REST4, REST5 and HBG (random) Guides respectively. The RT-qPCR experiments allow us to assess the amounts of each RNA isoform present in the H82 cells. We have carried out three selective amplifications for each of the 6 samples above:
- First Round of Amplification is selective Amplification for REST using primers spanning the alternative exon junction but not the exon itself.
- Second Round of amplification for REST4 is by using primers spanning the alternative exon.
- Third round of amplification is the selective amplification of Beta Actin gene as a housekeeping gene
We can see from these results that REST Guide 2 and REST Guide 4 performed the best.