RNA purification with TRI_ low yield - (Nov/30/2014 )

Hi!!!

I am having a lot of problems with RNA extraction using TRI reagent (Sigma). I know that this is a quite common topic but I couldn't find an answer to my problems, soif you have time to read and reply I'll be very thankful!

I followed the protocol of my own lab which is identical of that of Sigma, except that I spin at 13.000g instead of 12.000 g and after adding 70% cold ethanol I just spin 30 sec at top speed at RT (instead of 5 min at 7.5g in the cold room). The first time it worked fine, I got 5 microg/microL from 5 x 10^6 cells, 260/280 was 2.01, 260/230 was 1.53. I repeated it with 1x10^6 cells, the yield was around 2, but the 260/230 was worse, between 0.8 and 1.4 depending on the sample. From the 3rd time I always got awful 260/230 ratio, even 0.01, so I decided to try different protocols. I spin at 12.000g and I added 2 extra washing with 70% cold ethanol and it worked good for big samples (5x 10^6 cells), with a yield of 4.3 microg/microL and a 260/230 ratio of 2. I tried also the Sigma protocol (http://www.sigmaaldrich.com/technical-documents/protocols/biology/tri-reagent.html), repeating twice the last washing with 75% cold ethanol and the spin at 7.5g in the cold room, obtaining a quite good 260/230 ratio (near 1.5) but an extremely low yield, aroung 0.5 microg/microL for 1x10^6 cells.

Today I repeated both Sigma and lab protocols with extra washing trying to increase the yield by pipetting more (60 times, 2-3 min) and increasing the incubation time (10 min after adding TRI; 15 min after adding choloroform, 10 min after adding isopropanol) and I've got increadibly low yields! 1.7 microg/microL for 5x10^6 cells (Sigma protocol), and 0.4 microg/microL for 1x10^6 cells (lab protocol). I had a clear pellet, I'm sure I didn't touch it when I removed the supernatant and I resuspended it in 10 microL water pipetting 40 times, flicking 20 times and briefly vortexing.

With the old protocol I usually got between 1.7 and 2.5 microg/microL, it was contaminated with pheno (peaks at 230 and at 270 nm), but the yields was still enough for our purposes, only once I got 0.4 microg/microL for 1 million cells. I really don't know how to solve these problems! And I need to extract RNA from samples of 1 million cells and even less (FACS sorted)! :(

Note: The first 2 times that I did the extraction I didn't respect the timing strictly, as I was busy and it happened that I left the samples in the centrifuge in the cold room for 15-20 minutes after the centrifugation with isopropanol. I remeber that when I've got the low yield with old protocol I left the samples in the cold room for ca 20 min before the centrifugation with isopropanol, and today I left them in the cold room 15 min after the centrifugation with choloroform. It seems me odd that a time incubation in cold room before isopropanol centrifugation might affect the yield (and indeed the pellet is big and clear at the end), and my supervisor agrees with me, but do you think that this might be the case? Incubation in cold room at some point before isopropanol centrifugation might affect the yield?!?!

I did these trials with 3T3 cells, I read that the expected yield with this method is around 5 microg/microL per million cells, so even in the first extraction the yield that I've got is too low?

Any advice and comment is welcomed!

Thanks in advance!

A new piece of the puzzle. RNA extraction from 3 samples of exactly the same amount of cells (a mother colture has been divided in 3). After resuspension (pellets has been left in water for 15 min to soak the water, then it has been pipetted for 1-2 min, vortexed and briefly spun to collect all the drops) the RNA solutions have been measured in a 1:10 dilution (0.3 microL of RNA solution in 2.7 microL of pure water, pipetted, vortexed and spun 2 sec). The same dilutions have been repeated 4 times, for a total of 4 measurement of 1:10 dilution for each of the 3 RNA solutions. Nanodrop measured totally different concentrations not only among the different samples, but also in different dilutions of the same samples and even between 2 drops of 1 microL from the same dilution. The differences varies from 3 to 10 folds, and there is no proportion between the samples. Run into a glyoxal gels, RNA amount is consisntent with the different measurements. The 260/230 ration in those preparations were between 0.22 and 1.5. depending on the dilution. Do you think that pheno and salts might affect the homogeneity of the solution even in 3 microL? a 260/230 ratio of 1.5 indicate an amount of pheno which might affect the homogeneity?

I didn't permformed a extra wash with EtOh in these preparations.

Thank you!