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RNA miniprep protocol - Blood

  1. Transfer 1-3 ml of whole blood (collected in heparinized or EDTA-treated tubes) into a 15 ml conical tube. Add 3 volume Red Blood Cell Lysis Solution and mix the solution by inversing the tube 5 times.

  2. Centrifuge the blood sample at 3,000 rpm (400 x g) for 5 min at 4oC. Remove the supernatant by carefully pipetting from the top of the sample. Do not disturb the precipitated blood cell pellet.

  3. Transfer 500 µl Buffer LY to the leukocytes pellets and vortex the solution for 1 min. Ensure that b-mercaptoethanol has been added before use.

  4.  Add 0.5 volume absolute ethanol (96-100%) to the lysate.

  5. Transfer the solution into a RNA column and centrifuge at 14,000 rpm for 1 min. Discard the collection tube with the flow-through and put the column back to a new collection tube.

  6. Add 400 µl RNA Wash Buffer to the column. Centrifuge at 14,000 rpm for 1 min. Discard the flow-through. Ensure that ethanol has been added to RNA Wash Buffer before use.

  7. Optional: Add 50 µl DNase I (5URNase-free) solution onto the middle of the column and incubate at room temperature for 15 min. Add 200 µl DNase Stop Buffer onto the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through. Add 300 µl RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through. 

  8. Add 400 µl RNA Wash Buffer to the column. Centrifuge at 14,000 rpm for 30 s. Discard the flow-through.

  9. Centrifuge the column at 14,000 rpm for 1 min. Discard the flow-through.

  10. Centrifuge the column with the lid open at 14,000 rpm for 1 min. This step is critical to remove residual ethanol for optimal elution.

  11. Place the column in a RNase-free 1.5 ml tube, add 50-100 µl DEPC-treated water to the column. Centrifuge at 14,000 rpm for 2 min. The RNA is in the flow-through liquid. Store the RNA solution at -20oC. 

  • Red Blood Cell Lysis Solution: 5 mM MgCl2, 10 mM NaCl, 10 mM Tris-HCl (pH 7.0) 

  • Note: It is highly recommended that RNA quality be determined before downstream applications. The quality of RNA can be assessed by denatured agarose gel electrophoresis with the ethidium bromide staining. Several sharp bands should appear on the gel including 28S and 18S ribosomal RNA bands as well as certain populations of mRNA and bands. If these bands smear towards lower molecular weight RNAs, then the RNA has undergone major degradation during preparation, handling or storage, RNA molecule less than 200 bases in length do not efficiently bind to the RNA column. An A260/A280 ratio of 1.8-2.0 corresponds to 90-100% pure nucleic acid.

 
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