Guidelines for RNA Purification from Whole Blood

  1. The Monarch Total RNA Miniprep Kit (NEB #T2010) can be used to process both fresh and frozen blood.

  2. Using the standard protocol, up to 200 µl blood can be processed. The protocol can be modified so that up to 3 ml of blood (or 1 ml of rodent blood) can be processed on a single column with appropriate scaling of reagents and reloading of the column.

  3. For rodent whole blood, the maximum sample input amount is 1 ml.

  4. For processing whole blood, ensure that you use the Monarch DNA/RNA Protection Reagent (NEB #T2011) as a 2X concentrate.

  5. When using frozen blood that has not been stored in a stabilization reagent, quickly thaw at room temperature in the presence of 2X Monarch DNA/RNA Protection Reagent (with vortexing or mixing) as indicated in the protocol.

  6. For frozen whole blood previously stored in DNA/RNA Protection Reagent, allow sample to equilibrate to room temperature prior to processing.

  7. After mixing whole blood with DNA/RNA Protection Reagent, perform all subsequent steps at room temperature.

  8. RNA Lysis Buffer is not used in the Whole Blood Protocol. Whole blood samples are lysed upon mixing with the supplied 2X Monarch DNA/RNA Protection Reagent concentrate.

  9. Isopropanol is added to the blood sample mixture after Proteinase K treatment, creating favorable binding conditions for RNA binding to the RNA Purification Column.

  10. You will skip the gDNA Removal Column step and the addition of ethanol to the flow-through, and instead load the isopropanol-sample mixture directly onto the RNA Purification Column. An optional on-column DNase I treatment is recommended to remove genomic DNA.


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