TRIZOL trouble - (May/02/2013 )
I've been using trizol to extract RNA for quite some time, and I believe I have gotten fairly good at RNA extractions...until this morning. I use Qiazol from qiagen (while some may say trizol is just trizol, there may be differences between companies of a proprietary nature). I do the following steps:
Samples (macrophage primary cells) have been lysed/homogenized in trizol, samples can be stored in -80C until further use.
I take out the samples, thaw them on ice. Once completely thawed, I add 200uL CHCl3 per ml of trizol sample to each tube. I shake the samples for 15-20 seconds, and the phase seperation step works fine for me. CFG 20 min 13,200 rpm, 4C (we have a "fridge" CFG). Then I take the aqueous phase (top layer) off (no more then 350ul so there is extra and I'm less at risk to get the interphase that way), add 1uL glycogen (carrier) then add 500uL isopropanol to precipitate the RNA). Shake tubes, then 10 minutes room temperature, sometimes 1hour@ 4C or -20C if I need to get lunch/dinner. I spin the tubes down for 20 minutes at 13,500 rpm to pellet the RNA.
I see the pellet. I have visibly seen the pellet multiple times this past week before, during, after adding 70-75% ethanol in DepC Water and storing it at -80C overnight.
I can't find the pellets. I'm not sure what I'm missing, or if I'm missing anything at all, but I could use all the help I can get (salvaging samples, re-extraction possibilities, RNA rescue)
Frustrated RNA-extracting person...
I never keep the lysate in Trizol at -80C. I always keep my sample in RNA Later, and then if I want to extract RNA I will take the sample out of the RNA Later and extract with Trizol. We also don't use glycogen, and don't keep in iso p for 1 hr. it is very difficult to see RNA pellet. it is usually a transparent pellet, unlike DNA pellet. have you checked your concentration?
I've used a similar method previously to extract RNA from zebrafish tissue and it's worked fine, although I don't keep my RNA in 70-75% ethanol overnight at -80oC. Did you do that before? And did you stop at this step because you no longer saw the pellet? Or did you carry on and found no RNA left at the very end?