No RNA from cell pellet using Qiagen MiniKit!!??!!
Posted 13 May 2009 - 01:10 PM
Posted 14 May 2009 - 09:03 PM
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.
Posted 18 May 2009 - 03:37 AM
Posted 06 November 2009 - 12:40 AM
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?
Posted 26 March 2010 - 11:18 AM
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.
Edited by AngusWilliam, 26 March 2010 - 11:20 AM.
Posted 30 March 2010 - 04:46 AM
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.
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