Monday, June 8, 2009

RNA Extraction From Fresh Blood

RNA can be extracted from blood since whole blood contains nucleated white cells that constitute an easily accessible source. RNA extraction from blood will be more successful if the nucleated white cells are first isolated from the red cells since the red cells are a rich source of ribonucleases that are able to degrade RNA. It is important to minimize degradation by following the appropriate recommendations for handling RNA. The methods of RNA extraction usually comprises of three steps which are cell lysis, partitioning of RNA into a solvent fraction, and recovery of RNA from the solvent by precipitation.

Here are the materials that you need:
  1. X Phosphate-buffered saline (PBS).
  2. Lymphoprep (Nycomed).
  3. 10-mL centrifuge tubes (polypropylene or glass).
  4. RNAzol B (Note: RNAzol B contains guanidinium thiocyanate which is an irritant, and phenol which is a poison. It is therefore recommended that RNAzol B is handled in a fume cupboard.).
  5. Chloroform.
  6. Isopropanol.
  7. Ethanol.
  8. 5 M sodium chloride.

And The Method is:
  1. Dilute 10 mL of anti coagulated whole blood 1:2 with 1X PBS in a sterile plastic 20 mL universal.

    A suitable antrcoagulant is 3.8% trisodmm citrate diluted 1: 10 mto whole blood.

  2. Carefully layer 10 mL of the diluted blood onto 3 mL of lymphoprep in each of 2 x 10 mL polypropylene or glass tubes able to withstand centrifugation at 800g. Ensure that a sharp interface is obtained with little or no mixing between the blood and separation fluid.
  3. Centrifuge at 400g for 30-40 mm or 800 g for 15 min at room temperature.

    g-Force can be converted into rpm using the formula g = 0.0000118 x r x N2, where r = radius in cm and N = rpm.

  4. Following centrifugation, a clear solution IS obtained with aggregated erythrocytes sedimented to the bottom of the tube. Mononuclear cells, including lymphocytes form a distinct, cloudy band within the clear solution at the interphase of the upper sample plasma layer and the lower Lymphoprep solution (see Fig. 1) Transfer the mononuclear cell layer to a separate tube using a pipet tip or Pasteur pipet. The upper layer may first be removed to just above the band, if desired.
  5. Make the cell solution up to 5 mL with 1X PBS, invert to mix and centrifuge as in step 3.
  6. Decant the supernatant. The lymphocyte pellet may be stored for several days in this condition at -20oC before subsequent processing if desired.
  7. Lyse the cells by the addition of 0.5 mL RNAzol B Solubilize the RNA by passing the lysate through the pipet a few times.
  8. Transfer the lysate to a sterile Eppendorf, add 50 microliter of chloroform, shake samples vigorously for 15 s, and incubate on ice (or at 4oC) for 5 min. (Samples can be stored in this state for 1-2 h).
  9. Centrifuge the suspension at 12,000 g for 15 min in a microfuge.

    Ideally, microfuge spins from this stage onwards should be carried out at 4oC but may be performed at room temperature if a refrigerated micromge is not available.

  10. Transfer the upper aqueous phase (carefully avoiding the interphase, which contains DNA and proteins) to a fresh Eppendorf, add an equal volume of isopropanol (approx 400 microliter), and store for 15 min at 4oC (or at -20oC overnight).
  11. Microfuge samples at 12,000 g for 15 min. The RNA pellet should be visible at the bottom of the tube.
  12. Remove the supernatant and wash the RNA pellet once by adding 800 microliter of 75% ethanol, vortex and centrifugation at 7500 g for 8 min.
  13. Resolubilize the RNA in 0.2M sodium chloride (e.g., 192 microliter water + 8 microliter 5 M sodium chloride). Precipitate sample with 400 microliter of 100% ethanol at -20oC for 1 h.
  14. Centrifuge and ethanol wash as above (steps 12 and 13).
  15. Allow the pellet to dry with tube open at room temperature for 15 min
  16. Solubilize the pellet in 50 – 100 microliter of DEPC-treated water. The sample may be heated to 52-60oC for 5-15 min to aid solubilization.
  17. Quantitate and analyze the RNA.


Reference: Number 16 on References