With a few additional reagents and significant protocol modifications, the RiboPure™-Blood Kit (Cat #AM1928) can be used to isolate total RNA that includes the small RNA fraction from RNA later® Solution-treated human blood samples. Using this modified protocol, total RNA that includes miRNA, tRNA, 5S/5.8SrRNA, and other small RNA species is recovered.
Before you start, heat >100 μL per sample of Elution Solution to ~75°C in an RNase-free tube.
- Pass the sample through a Filter Cartridge
Filter the sample through a Filter Cartridge using a vacuum manifold. Alternatively, sample can be centrifuged though a Filter Cartridge by successively applying 700 μL of sample, centrifuging for a few seconds to pass the liquid through, emptying the Collection Tube and repeating. Discard the filter flow-through.
- Wash filter with 700 μL [70% Ethanol/ 30%Denaturation
- Remove the Filter Cartridge from the vacuum manifold and transfer it to a 2 mL Collection Tube (included with the RiboPure-Blood Kit)
- Apply 700 μL [70% Ethanol/30% Denaturation Solution] to the Filter Cartridge and centrifuge for ~5–10 sec to pass the solution through the filter. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube.
- Wash filter with 2 x 700 μL [80% Ethanol/50 mM NaCl]
- Apply 700 μL [80% Ethanol/50 mM NaCl] to the Filter Cartridge and centrifuge for ~5–10 sec to pass the solution through the filter. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube
- Repeat with a second 700 μL aliquot of 80% Ethanol/50 mM NaCl.
- After discarding the flow-through from the last wash, replace the Filter Cartridge in the same Collection Tube and spin the assembly for 1 min to remove residual fluid from the filter.
- Elute RNA with 150 μL preheated Elution Solution
- Transfer the Filter Cartridge into a labeled Collection Tube (provided with the RiboPure-Blood Kit).
- Apply 150 μL Elution Solution (preheated to ~80°C) to the center of the filter, and close the cap. Leave the assembly at room temp for 1 min
- Centrifuge for ~20–30 sec at maximum speed to recover the RNA in the Collection Tube.