Immortalized cell RNA isolation
This protocol is a simplified version of the Qiagen RNeasy kit. Please read their book before attempting the basic protocol.
Cell growth and harvest:
Your cells should be nearly confluent when you harvest them. 6-well plates provide adequate RNA for multiple experiments.
1. Rinse with PBS at least once, being careful not to dislodge cells. Shake off all media and liquid.
2. Use a cell scraper to lift the cells. Work quickly to prevent them from drying out.
3. Quickly add the RLT buffer. For large numbers of cells, add 700 uL. Otherwise, add 350 uL.
4. Pipet the cell/RLT mixture to microfuge tubes. You can freeze at -80C at this point, or continue with the RNeasy kit. RNA can be stored indefinitely, although I find that after a year or two it does not work as well.
Follow the basic RNeasy protocol at this point. I strongly suggest using the optional RNase-free DNase add-on kit, which removes genomic DNA. This is particularly important if you will be using the SYBR green method. Alternatively, you could design your primers to overlap intron/exon sites, which theoretically should prevent the need for the DNase.