Hello all,
My name is Matt, and I am a 2nd year PhD student whose work currently focuses on intestinal gene expression. In brief, to collect intestinal tissue samples, we harvest sections of the intestine, flush the lumen with ice-cold 1X PBS, and remove as much fat from the outside of the intestine as we can. Finally, we "open up" the sections by cutting down the length of the sections, flatten them out on plastic Petri dish covers on ice, and scrape up the mucosa layer using glass cover slips. So, we don't collect the muscular layer of the intestines (besides any cells that may be incidentally scraped up), and do collect all cells of the mucosal layer (enterocytes, goblet cell, stem cells, resident immune cells) and any bacteria still adhered to the mucus layer.
The specific cells I am interested in, the stem and immune cells, represent a small fraction of the overall cell population, and I worry their low number means that when I isolate mRNA and perform RT-qPCR, changes in gene expression will get lost in the "noise" of all the the other cells' mRNA. I know that isolating cells of interest would be the best way to reduce noise, but right now a lot of my work is exploratory and as such faster/cheaper methods to generate pilot data is best. Here are my specific questions:
1) Besides following MIQE guidelines, are there any other helpful guidelines people could point me towards for primer design and optimization?
2) Our lab uses 18S as the reporter gene, and we use the ΔΔCT method for calculating relative expression. In previous experiments I confirmed 18S is actually the most stably-expressed reporter gene in a panel of 10 other genes I tried out, but should I use a second reporter gene and use the geometric mean method for calculating expression?
3) Any other general RT-qPCR tips/advice?
Thank you!