No RNA from cell pellet using Qiagen MiniKit!!??!! - (May/13/2009 )
I am trying to extract RNA from PBMC's (lymphocytes and monocytes) using Qiagen's RNeasy minikit, and I'm not getting ANYTHING. I isolated the PBMC's using Ficoll Paque Premium. Following Qiagen's instructions, I used about 3 million cells because I was unsure how much RNA this type of cell would have. I counted them using a hemocytometer and stained them with tryphan blue. I seperated them so there I ended up with 3 million larger WBC cells in each tube. I didn't take in to consideration all the small red blood cells in the count because that wasn't really our focus but looking back that could be the reason I'm not getting any RNA. Anyway, I tried 4 different times using the minikit. Different variations I tried was using 600ul lysis buffer instead of 350ul due to the abundance of RBC. I tried homogenization using a Qiashredder and also using a 20g needle. I also tried increasing the concentration of ethanol from 70% to 90% due to the residual media that remained when the supernatent was asperated. Everytime I run a nanodrop I don't even come close to what could be considered usable RNA. I know I'm doing the procedure correctly because I can successfully extract RNA from frozen tissues using the same methods. Somewhere, somehow, one of the columns (either the Qiashredder or the spin column) is getting clogged. Could anyone give me some insight as to why I'm having this problem. I was thinking that the RBC's could be clogging things up but 1)There were only ~2x more RBCs than WBCs so that would put the count to around 9 million and that isn't really unheard of for homogenization using a Qiashredder 2) I can succussfully isolate RNA from tissue which I would think would be harder to homogenize than cells.
when you say you don't get anything, do you mean low concentrations of degraded RNA or really nothing at all?
Did you use the stained cells for RNA extraction? I've never worked on blood cells, but am not sure about the effect of the stain on the RNA.
You could try without, and see.
ALso try to extract with trizol and then see if its an effect of the column.
Ahrenhase on May 14 2009, 07:10 AM said:
Thanks for your response. No I did not use stained cells for extraction, i just used a 10ul aliquot to count them. I'm with you though, I'm going to try a preliminary trizol extraction first. I think the cells are gumming up the column.
Ahrenhase on May 18 2009, 03:37 AM said:
Hey Ahrenase did you get it to work?! I have been purifying RNA from tumour cell lines and gettnig really nice quality RNA, good yields and really clear rRNA bands on agarose gel. But when trying from primary Tcells and B cells, as well as total PBMCs, purified using density centrifugation and MACS sorting, I get huge genomic DNA contamination and very little RNA even though I'm using exactly the same kit, method etc. So I was trying to see if anyone else had better success... how did yours go?
There are two problems you need to address (if you want to get some RNA from these cells)
1) Don't use so many cells.
3x10^6 cells is masses. 1x10^6 cells should provide more than enough RNA. Using too many cells tends to result in an incomplete lysis and clogged spin-filters (especially in the steps that use the RNA-binding-membrane filter). Clogged filters are a really bad thing for RNA: the trapped RNA just gets chewed up by RNases.
2) Lyse the RBCs. Try this protocol:
Protocol for isolating Leukocytes
Collect whole blood.
1) Spin down (1000g for 10 min, 4oC).
2) Resuspend cells in RBC lysis buffer (RBC lysis buffer: NH4Cl <150mM>, KHCO3 <10mM>, and EDTA <0.1mM>. pH value is 7.4) and place on ice for 10 minutes.
3) Spin down (1000g for 10 min, 4oC) and wash once using cold PBS.
4) Count cell number.
5) According to the cell number, add 600ul RLT lysis buffer per 1X10^7 cells (from Qiagen RNeasy mini kit, cat #.74104), and homogenize the lysate.
I strongly recommend using Superase-In or some other RNase inhibitor - put some in the catch tube before you wash the RNA off the spin-filter membrane.
You could also try using the RNAqueous 4-PCR kit, it's dope.
For every Dneasy or Rneasy user:
1. never throw away the columns until you got a concentration reading( if too much material it will clog the column and the nucleic acids are stuck in the column.
2. The wash step that is "optional" is really don't optional DO IT
3. The dry step that again the protocol say is optional do it also
4. Warm the elution buffer or water
5. Do the elution in two step (add half of the vol. incubate for 2 (DNA) or 5 min for RNA spin, add the other half incubate again, spin
6. If the concentration or yield is low add more elution buffer and warm the column spin.