For PBMCs isolation by Ficoll density gradient centrifugation, bring blood and reagents to room temperature before starting processing. Ideally separation should be performed as soon as possible after blood collection. Few hours delay would create no problem if blood is kept refrigerated. Further delay may cause hemolysis.
Use conventional methods for PBMCs storage, nothing special is required for PBMCs meant for RNA extraction, in my opinion. Use following questions for detailed discussions already present at ResearchGate:
https://www.researchgate.net/post/Optimal_conditions_for_freeze_storing_PBMCs
https://www.researchgate.net/post/PBMC_isolation_from_buffy_coat
Good luck!
Thanks, somebody suggested me to store cells in RNA Later for longer storage, will it be for any better?
RNA once extracted will have to be kept at -80 degrees and will keep well at this temperature. However certain commercial preparations may help in preventing degradation by any contaminating RNAses.
But inside intact PBMCs, your RNA will be naturally protected from RNAses and further at -80 degrees, degradation should not be an issue. So you don't need solutions like "RNA Later" if you have a -80 degree freezer available. Otherwise, you will need this type of solution to preserve cellular RNA in the absence of freezing.
If you are going to extract RNA anyway, you can lyse the cells in an RNA binding buffer that comes in number of commercial kits. Alternatively, you can suspend the PBMCs in Trizol. This protects the integrity of the RNA for long periods of storage. A last option is to suspend the cells in a solution of RNALater and store at -80C in suspension.
I have worked on extraction of viral RNA from cells infected with an RNA virus. PBMCs preservation for future extraction of mRNA for indigenous gene expression is a bit different. Mr. Warren Fiskus, have given right suggestions!
Thank you all, will share my experiences with you guys soon.
Thanks again
Dear Zaheer Nizamani. In your experience doesn't ficol rests disturb RNA isolation? what do you think of this: "RNA extraction with residual Ficoll contamination doesn't work. Hypothesise that Ficoll binds RNA and takes it to organic phase of extraction."
http://www.bio.net/bionet/mm/methods/1999-September/077927.html.
thank you !
Peter
Im having trouble about the RNA extraction from PBMCs, using many wells from the same sample could obstruct the filter from the column? Usually we put 3 to 4 wells together, and the RNA has been very low. Could someone have a effective protocol for it? thank you!
Hi Juliana,
I faced similar problem for long time, but no longer. Wash your cells with DPBS to remove any ECM from the suspension. Then after adding tryzol and homogeneizing, freeze them. Thaw on ice. Once thawed put them on a water bath at50-60 C for 10 min. Spin at 12,000 x g at 4 C for 15 min and recover the 80% supernantant. This will give you high quality and yeld RNA
Hi,
I am trying to extract RNA from stored PBMCs(stored in liq nitrogen). But basically i am getting blank gels after the entire procedure.I have thawed
PBMC in 10% FBS and after that tried with both trizol LS and TRI reagent.Anyone facing same problem?
what cryoprotectant are you using to freeze the cells? What is the composition of the freezing media. 10 % FBS seems very low... protocols going from 50-90% work better. You may be working on samples of dead cells with degraded nucleic acid.
If the purpose is to use them for RNA, I would use RNA lysis buffer (Tryzol, etc) immediatelly after cell isolation, making sure you are following the exact manufacturer recomendations...
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