RNA isolation - (Jan/25/2001 )
while extracting RNA, we always get low 260/280 ratio about 1.4-1.5 . is there any better way than repeating our protocol?
What protocol are you using? If you have enough money to buy a kit, I very highly recommend the Qiagen RNA/DNA maxi kit. It lets you extract whole RNA and Genomic DNA using the same protocol. Additionally, it lets you extract both small and large quantities of these nucleic acids (depending on how much tissue you want to use) I have used this kit many times. I have also screwed up many times (forgot to use B-mercaptoethanol once, accidentally added elution buffer at the wrong step, etc)- But even when I think I have screwed it up, I've gotten RNA with a 260/280 from 1.9-2.1 every time, usually right at 2.0.
Tell me more. What's your source of message, what's the method.
i would do an extra phenol/chloroform extraction followed by just a chloroform extraction. the chloroform extract. will remove any residual phenol which will alter the OD ratio. this is of course assuming thatyou followed the historical RNA isolation method as cited by Maniatis. followed up by ppt. overnight in the -20C
I'd like to ask you if it is possible to overcome DNA contamination during RNA isolation. I use the TRIzol method and I always have DNA contamination.
Thanks in advance
for qiagen kit, does it allows isolation of siRNA too? i'm intersted on that point...
On the trizol manual, it's told that low ratio 260/280 may be due to a bad resuspension of rna. Try increase the amount of DEPC water of our prep. Will be better. One other possibility is to heat your rna 10' at 65° before make a OD measure.
To Pon103 :
try increase the amount of trizol you use for cell/tissue lysis. It will reduce contamination possibilities...