SLX5 and Huntington’s Disease: Second Update

In my last blog post, I spoke about developing a protocol to verify findings from a new data set. This has been my focus since that update.

The existing data is a list of genes that the mutant Htt protein either up or down regulates, including the reversal of these effects by the addition of an extra copy of the SLX5 gene. In order to verify these findings, I have chosen five of the genes listed to test myself, in an attempt to obtain the same results.

To do this, I am running qPCR reactions on cDNA made from yeast strains with and without the extra copy of SLX5, and in five different reactions, will test for each of the five genes. This is a lot of jargon, but I am trying to prove that mutant Htt leads to more or less transcription of a certain gene, and extra SLX5 reduces this. The result of the transcription of a gene is mRNA made complimentary to the strand of DNA carrying the gene. Therefore, the relative amount of transcription can be quantified by the amount of RNA that exists for that particular gene. This is not measured directly, but rather a reaction is run using reverse transcriptase, which makes DNA, specifically called cDNA, using the RNA as a template. Then, a qPCR reaction is run, which quantifies the amount of your specific gene in the cDNA using probes which only can adhere to the portion of cDNA you are looking for.

Using this process, I have obtained data for each of the five genes. However, the data is not as tight or clear as is ideal, so I have continued running the reactions to get more exact results. I will continue to refine the process until I get the best data possible.