problem with Trizol RNA extraction
Posted 23 July 2010 - 05:58 AM
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
Posted 24 July 2010 - 01:15 AM
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 ???
Posted 25 July 2010 - 02:46 PM
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?
Posted 26 July 2010 - 08:17 AM
Posted 26 July 2010 - 03:49 PM
Posted 27 July 2010 - 11:24 AM
Posted 01 August 2010 - 11:47 AM
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
Posted 02 August 2010 - 07:25 AM
then heaven will be yours, before you meet your end