I started off the morning talking to Erin and Diego (a graduate student) about the electrophoresis chips. These chips are the most costly things that I use in the lab. Recently, Diego and I had been getting slightly messed up results with the chips. So Diego, Erin, and I all ran a chip together to make sure that our procedure in setting up the chip was proper. For the first time in a while, our data had no issues. However, there is only 1 chip in the entire lab left, so I cannot get new data from the PCR that I've run until we get more.
So, with the free time I had, I started calculating values and putting them into an excel sheet. When I looked at the data I had, I got a little worried. The cell line treated with siRNA was only 400 less than th control (treated with nothing) cell line, while the cell line treated with only the Dharm was nearly 100 higher than both of the other values. I went to Erin to see what I had done wrong.
She told me I hadn't done anything wrong. As I walked into her office, I realized that I left out a factor of ten in my control value. This made sense as the siRNA should silence the gene expression by over 70% (which in the case it did). Two of my data points made sense, but the Dharm sample still confused me. This was a result of my own lack of understanding.
Erin told me that when they use siRNA, they use the Dharm to get the siRNA into the cell. The cell membrane is hydrophobic, and the siRNA is hydrophilic, so the cell membrane (neutrally charged) does not want to let the siRNA (negatively charged) into the cell easily. The Dharm effect allows for the siRNA to pass into the cell through endocytosis by using liposomes. So all the Dharm-treated-only cells had were liposomes inside them. Erin then told me that the liposomes affect gene expression in both directions (higher and lower). As long as my data differences weren't too drastic (over a 1000 fold difference) that my data was still fine. Thus, I finally have data on all 3 of the cDNAs. I will start getting more data for CEBPG and for another gene, ERCC5, when we get more electrophoresis chips.
Who pays for the chips you use? How much do they cost? Where do they come from? What do they do?
ReplyDeleteDoctor Willey's lab purchases the DNA chips from Agilent Technologies. They cost $340 for 25 chips, so we really don't like wasting them. Usually we try to fill every chip before running it. There are 16 wells on a DNA chip: 12 for samples, 3 for the gel mix, and 1 for the ladder. A machine made to read the chips sucks in all of the reagents and uses electrophoresis to see how large and how many molecules there are relatively to the calibrating ladder molecules.
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