RNA extraction using qiagen RNeasy mini kit - (Nov/03/2008 )
Hi,
I am trying to extract mRNA out of different tissue samples of mouse using qiagen RNeasy protect mini kit. However, I am not getting the concentrations that they say one should get. I am using 10mg or less of tissue as advised and performing everything as stated. I homogenise my tissue using mikro dismembrator. Lately I lost RNA for thymus and for others also I have got about 20, 30,40 etc ng/ul of RNA. THe quality is ok between 1.9 to 2.2.
Can anybody please suggest some tips to increase the yield?
Thanks a lot
Though I'm not familiar with your 'Mikro Dismembrator', A couple of procedural modificatons I've adopted come to mind....
1) If you're not using b-ME in the RLT extraction buffer, you should. According to their protocol it's optional, but in my brief experience trying w/o b-ME, my yield was way lower than expected.
2) Give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer)
3) Make sure the EtOH is completely evaporated off of the column prior to elution. The manual says to leave the cap open for 1 minute, I think. I leave it open for 5 min at 60`C.
4) Use water to elute the RNA that is warmed to ~60`C. I elute with 50ul, spin, take the eluate, and run it through the column again, spin, and then add 50 ul of fresh water and spin one last time. You'll yield ~97ul of (total) dilute eluate that can be concentrated to 10ul in a spin-vac to should give you a pretty decent concentration for most down-stream applications.
Good Luck!JAH
1) If you're not using b-ME in the RLT extraction buffer, you should. According to their protocol it's optional, but in my brief experience trying w/o b-ME, my yield was way lower than expected.
2) Give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer)
3) Make sure the EtOH is completely evaporated off of the column prior to elution. The manual says to leave the cap open for 1 minute, I think. I leave it open for 5 min at 60`C.
4) Use water to elute the RNA that is warmed to ~60`C. I elute with 50ul, spin, take the eluate, and run it through the column again, spin, and then add 50 ul of fresh water and spin one last time. You'll yield ~97ul of (total) dilute eluate that can be concentrated to 10ul in a spin-vac to should give you a pretty decent concentration for most down-stream applications.
Good Luck!JAH
Hi,
Thats very helpful. I was washing once with RW1 and twice with RPE. I am using DTT instead of 2-me due to restrictions. I usually homogenise the tissue with a ball bearing in a small enclosed vessel. The machine gives it a high rpm, ofcourse the tissue,buffer RLT ball bearing and vessel are liq nitrogen cooled. For elution procedure you suggested, i dont think we have a spin-vac and am not very familiar with it (1st year phd student!). Could you please give any alternatives. Thanks a lot for your suggestions.
We switched recently from the Qiagen RNeasy kit to using Trizol reagent for RNA isolation, mainly to save money. An unexpected side benefit was an increase in yield of RNA (for similar number of adherent cells, compared to the RNeasy results). Trizol RNA isolation takes a little longer though.
With the RNeasy kit we usually got around 150 ng/microliter from 300,000 HeLa cells, using Trizol we get around 225 ng/microliter pretty consistently. Quality, both 260/230 and 260/280, have been good with either method when the protocols are followed carefully.
Haven't worked with tissue samples though. Were they stored properly? RNA degradation could be an issue.
You might get more looks and subsequently more advice posting this in the molecular biology page.
Depending on the type of tube, try using a low-bind tube to prevent RNA from sticking to the plastic. I have found that this helps increase my RNA yeild. Also, if you are looking to store it, I found this product that is pretty good. Its called RNAstable from a company called Biomatrica. Its room temperature storage. I have tested it on my RNA, and it has lasted longer than samples I have kept at -20C and -80C. Just a suggestion, and hope this helps.
Austin