we spin down the sample tube, take the buffy coat, lyse the red blood cells and trizol extract.

7.1. Isolation of Cells from whole blood or bone marrow for RNA Extraction

7.1.1. Spin blood tubes in clinical centrifuge at ~800g for 5 minutes. For highest quality RNA this step should be initiated as close to draw time as possible. Prepare a 15ml conical tube for each sample with 14ml of 1X Red Blood Cell Lysis (RBCL) Buffer..

7.1.2. Remove white cell layer from spun tube with a transfer pipet and add to appropriate 15ml tube. Invert tube several times to thoroughly mix. Let the tube sit at room temperature for 5 minutes. The red blood cells will lyse during this time.

a. Some RBCs will be transferred with the WBC layer. These will lyse in the RBCL Buffer. If there is little or no visible WBC layer, take the top of the RBC layer to the 15ml tube, immature WBCs can be in the top layer of RBCs.

b. After 5 minutes, if the sample still appears cloudy, let the lysis tube sit at room temperature an additional 5 minutes.

7.1.3. The sample should now look transparent.

7.1.4. Spin the lysis tubes in the clinical centrifuge at ~800g for 5 minutes.

7.1.5. Pour off supernatant in sink being careful that the cell pellet does not slide. Unlysed RBCs may be with the pellet. Unless there are a large number of them, there will be no problem. If unsure about the number of RBCs, lysis can be performed again. Add 1ml 1X RBCL Buffer, gently resuspend pellet and bring volume to 15 ml. Let sit at room temperature again and respin. Again, decant supernatant carefully. Invert tubes to remove liquid. (Leave a small amount of liquid proportional to the size of the cell pellet as you will briefly “flick” the tube to resuspend pellet. This facilitates pellet lysis in the next step.)

7.1.6. Take tubes to RNA bench. Add 1ml Trizol (a mono-phasic solution of phenol and guanidine isothiocyanate) to the 15ml tube and slowly pipet until the cells lyse completely. Larger pellets may require addition of more Trizol – always in 1ml increments.

a. If you have a very large cell pellet, work the cells into solution with the volume of liquid not poured off and remove a proportion with a transfer pipet

b. Larger pellets may require 2 or more ml of TRIzol Reagent. It is best to add the full volume of TRIzol to the pellet at the same time, as the lysed cells become so viscous that even distribution cannot be achieved once the cells are fully lysed. An overly viscous sample can lead to protein and/or DNA contamination in the RNA, and may require re-extraction. Due to centrifuge requirements, samples with 2 or more ml TRIzol will be aliquotted 1ml to each prelabeled tube. The RNA extraction can proceed with one or more ml of TRIzol lysed tubes.

7.1.7. Transfer the viscous Trizol/cell solution to labeled 2ml flat bottomed Sarstedt tube(s).

7.1.8. The lysed cells may be stored at –20°C to await RNA extraction.

The trizol protocol is pretty standard.

3.2.1. Red Blood Cell Lysis Buffer, 10X

500mL reagent [final]

44.95g NH4Cl 1.68M

5.0g KHCO3 0.1M

100uL 0.5 M EDTA 0.1mM

Dissolve reagents in 400 mL of CLRW. Adjust pH to 7.3 and QS to 500 mL.

Stable for 12 months at +4C.

Note that the RBCL Buffer recipe is 10X.

Good luck

Given that PBMCs are some of the most transcriptionally active cells in whole blood I would start with them.

Everything that Pamela said above is spot on, but if you want to look at PBMCs add the following at the beginning (due to the amount of RBCs present):

1. A ficoll separation.

2. Several washes with RPMI1640 or similar (utilize patient plasma as a replacement for FBS if you have enough)

3. Treatment with 1x RBCL buffer for 3-5 minutes followed by 2 additional washes (50 mL).

4. Move on to RNA isolation.

Not sure what profile you're looking for with the RNA, but I don't think ficoll will effect them too much. I attached pdf with a good overview of RNA isolation from blood.

Blood is collected in standard EDTA Vacutainer® Tubes. A vented transfer spike is inserted into this tube and attached to the filter device (See sidebar, Simple Leukocyte Filtration from Whole Blood). The assembly is then connected to an empty 10 ml evacuated blood collection tube, which provides the suction needed to draw the blood through the filter without opening the tube. A 9–10 ml blood sample can be filtered in about 2 minutes. The filter is then flushed with PBS to eliminate residual red blood cells and with RNAlater to stabilize the RNA in the intact, captured cells. The entire process of leukocyte fractionation and stabilization requires less than 5 minutes and is free from centrifugation and other time-intensive steps that may perturb mRNA expression profiles. The RNAlater-treated filters can be stored at room temperature for several days or at -20°C for months with no loss in RNA integrity

please protocol available on this link

http://www.lifetechnologies.com/uk/en/home/references/ambion-tech-support/rna-isolation/tech-notes/isolate-rna-from-white-blood-cells-captured-by-a-novel-filter-sy.html

Thought about using density gradient centrifugations? Might help tidy up your cell populations.

Few cool papers

http://www.sciencedirect.com/science/article/pii/0022175974901094

http://informahealthcare.com/doi/abs/10.3109/08820137209022965

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC302448/

Thank you very much. I'll try your suggestions!

Hi Reyhaneh,

I agree with everyone's protocols. However, to simplify the isolation of PBMCs I suggest (if available) to use BD vacutainer CPT cell preparation tubes. All that is needed is to collect blood into the CPT tube, centrifuge and the WBC sit on top of a "plug". Then carefully remove the rubber stopper and collect the patient plasma and then collect the WBC and proceed as above for RNA isolation. The tube are more expensive, but the simplicity and time saved is worth the added expense.

Hi Daniel Medina,

Thanks for suggestion. Let me Know that you use Ficoll separating solution for isolation of live peripheral blood lymphocytes.

Thanks again.

Dear Daniel

I think this solution is suitable for WBC isolation, What is your idea?

Ficoll is fine. i use it all the time for routine studies. When I want the plasma and the WBC or time is limiting I like to use the CPT tubes.

Dear Daniel

Thank you for letting me know.

Try to isolate by using histopaque (sigma) method.

It will give you good quantity of cells and avoid any interference of RBC.

After that add trizol reagent to wbc and keep those for overnight at 4 degree cel.

At next day, try to isolate RNA using Trizol method or RNeasy kit (quigen)

Dear Sushant Shengule

Thanks for your suggestion.