Guidelines for RNA Purification from Whole Blood
- The Monarch Total RNA Miniprep Kit (NEB #T2010) can be used to process both fresh and frozen blood.
- 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.
- For rodent whole blood, the maximum sample input amount is 1 ml.
- For processing whole blood, ensure that you use the Monarch DNA/RNA Protection Reagent (NEB #T2011) as a 2X concentrate.
- 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.
- For frozen whole blood previously stored in DNA/RNA Protection Reagent, allow sample to equilibrate to room temperature prior to processing.
- After mixing whole blood with DNA/RNA Protection Reagent, perform all subsequent steps at room temperature.
- 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.
- Isopropanol is added to the blood sample mixture after Proteinase K treatment, creating favorable binding conditions for RNA binding to the RNA Purification Column.
- 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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