Blog Post #2: Describe an obstacle that you overcame during your internship.

My lab uses RT-qPCR to study gene expression.  To use this technology, we extract mRNA from cells or tissue (depending on the experiment), use RT-PCR to produce cDNA from it, and then use qPCR to determine the relative amounts of each transcript we are interested in.  This requires a ‘housekeeping’ gene, which basically tells you the amount of cDNA that’s present in each sample.  We subtract the amount of housekeeping gene that is present from the amount of our gene of interest that is present to normalize the data before analyzing it.  We use Taqman probes to specify which gene we want to quantify in each replicate.

We recently discovered that the Taqman probe for our normal housekeeping gene, GAPDH, targeted not only GAPDH, but also over 20 other sites in the genome.  This is problematic because these other sites may be affected by the different treatments and genotypes we use in our experiments.

Last week, another lab member and I identified three candidate housekeeping genes whose Taqman probes were specific to that gene.  We did this by comparing several papers that identified good potential housekeeping genes and cross-referencing them with our own RNA-seq dataset, which tells us the level of each transcript in normal mice, mice heterozygous for a mutant allele of the gene we study, and mice homozygous for a mutant allele of the gene we study.

This week, I performed several RT-qPCR experiments and determined which of the three was the best housekeeping gene.  Now we can repeat our old experiments to make sure our results are dependent only on our experimental conditions!

 

I’ve attached an image of myself with another LSA intern in the lab.

 

 

 

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