7 replies to this topic

[Annabell S.]

Hi all,

I have brain tissue that I'm trying to extract RNA from. I added 2 mL of Trizol and 400 uL of chloroform, vortexed, and centrifuged at 4C for 15 mins. After centrifugation, I could not detect an interphase layer in the tube; all I saw was a top aqueous layer and a bottom trizol layer. I anyway extracted this top layer and continued with the RNA purification. However, in the end I got no RNA from this tube. Does this mean that the cells did not lyse properly? And if so, what were the contents of the top aqueous layer?

Thanks for any advice

[nightingale]

hello,

haw u have this tissue .. ?
i mean, is it embedded in paraffin ???
are you sure you took "tissue" or was it more paraffin ???

how did u know there was no RNA ???
have you checked the two bands 18S and 28S on the gel ???

[bob1]

Did you check that the tissue was dissolved/homogenised before starting the chloroform step?  If not, it is very likely that you got no RNA because the cells weren't lysed.

How was the tissue handled? - added to trizol straight after taking out of the animal/patient? Snap frozen and stored at -80? etc?

2 ml of trizol is quite a lot, how much tissue are you using?

[Annabell S.]

Thanks for the replies. The tissue was snap frozen and kept at -80C. By eyeballing the tissue I thought it required 2 mL of trizol; there was quite a lot of sample. And I used the nanodrop, which indicated no RNA.. If the cells did not lyse, is there something I could do to recover the RNA?

[bob1]

If the tissue wasn't lysed properly there would have been big lump(s) left in the tube.  Snap freezing at -80 should be fine, brain is relatively low density, so amount is hard to estimate by eye, you may have used too much trizol, but you should still have got some RNA out the end.  Try running a few microlitres out on a gel to see if you can see the 18 and 28 sRNA bands.

[BryanC]

Did you do any type of mechanical homogenization or did you just add some Trizol and let it incubate?

[Annabell S.]

BryanC, on 27 July 2010 - 11:24 AM, said:

Did you do any type of mechanical homogenization or did you just add some Trizol and let it incubate?

I used a glass teflon homogenizer

[gfischer]

How well cleaned was the homogenizer?  RNAse contamination from the homogenizer could cause your yields to crap out.