RNA II

We start the protocol by freezing our samples immediately in liquid nitrogen to solidify the transcript that we are going to capture, this also disrupt any RNAse activity that could potentially degrade our transcript. We then grind our frozen tissue a in a frozen mortar with an RNA extraction/lysis buffer. We add this slurry of tissue/buffer into a homogenization column and centrifuge to filter through the RNA rich liquid we are after. Next comes the precipitation of RNA. This is facilitated by absolute ethanol. We add the ethanol/buffer+RNA mixture to a separate column that has high binding affinity to RNA. The RNA precipitates after a short spin and binds to the column matrix. We then add several wash buffers to “clean up” our sample that is soon to be eluted.  The second wash buffer contains ethanol that has been diluted to 70%. This allows for both ethanol and water soluble contaminants to be discarded. While isolating RNA, there is always genomic DNA contamination. This occurs due to shared biochemical properties between the two molecules. I prefer to do an “on column” RNAse free DNAse treatment that supposedly degrades 99.99% of contaminating genomic DNA. After this step, we are ready to elute our clean, total RNA. I use a spectrophotometer to determine the quality and quantity of my extraction.