Total RNA extraction from frozen placenta tissue
Scott Lindsay-Hewett
Abstract
This protocol describes the isolation of high-quality total RNA from frozen placenta tissue. Tissue is disrupted using a bead beater, and total RNA is isolated using the mir Vana miRNA Isolation Kit from Ambion.
Written steps are adapted from Ambion's manual for the mir Vana miRNA Isolation Kit and BioSpec's instructions for the Mini-BeadBeater-16.
Before start
Steps
Preparation
Clean workspace, pipettes, and gloves with RNaseZAP.
Prepare bucket of ice.
Heat nuclease-free water to 95°C.
Pre-cool microcentrifuge to 4°C.
Add 200 proof pure ethanol to mir Vana wash buffers as instructed.
Pull frozen placenta samples (~150-200 mg each) from -80°C and place on dry ice until ready to process.
Cell lysis and tissue disruption
Add approximately 1mL of 1.0mm zirconia/silica beads to each frozen placenta sample, and 700µL mir Vana Lysis/Binding Buffer.
Load samples immediately into Mini-BeadBeater-16 vial holder ring. Up to 16 samples can be accommodated.
Switch on the Mini-BeadBeater-16 and run for 0h 2m 0s.
Place samples immediately into pre-chilled microcentrifuge and spin for 0h 5m 0s.
Remove 500µL lysate, being careful not to draw up particulates, and dispense into a fresh labeled microcentrifuge tube.
Organic extraction
Add 50µL mir Vana miRNA Homogenate Additive (1:10 volume of original lysate) and vortex to mix. Incubate 0h 10m 0s``On ice.
Add 500µL Acid-Phenol:Chloroform (1:1 volume of original lysate) and vortex 0h 1m 0s to homogenize sample.
Spin 0h 5m 0s in pre-chilled microcentrifuge.
Carefully remove 350µL of the top aqueous phase and transfer to a fresh labeled microcentrifuge tube.
Total RNA isolation
Add 437.5µL (1.25 volumes) 200 proof pure ethanol and mix thoroughly.
Transfer up to 700µL lysate/ethanol mixture to mir Vana Filter Cartridge (placed into mir Vana Collection Tube) and spin 10000x g,4°C in pre-chilled microcentrifuge. Discard flow-through. Repeat with remaining volume of lysate/ethanol mixture.
Add 700µL mir Vana Wash Solution 1 and spin 10000x g,4°C in pre-chilled microcentrifuge. Discard flow-through.
Add 500µL mir Vana Wash Solution 2/3 and spin 10000x g,4°C in pre-chilled microcentrifuge. Discard flow-through. Repeat wash step.
After discarding flow-through from the last step, spin 10000x g,4°C in pre-chilled microcentrifuge to remove residual Wash Solution.
Transfer mir Vana Filter Cartridge into a fresh mir Vana Collection Tube. Add 40µL pre-heated nuclease-free water to the center of the filter and close the cap. Incubate 0h 1m 0s and then spin 0h 0m 30s in pre-chilled centrifuge to elute RNA.
Transfer eluate from the mir Vana Collection Tube to a low bind microcentrifuge tube and store at -80°C.
Quality control
Quantitate RNA using NanoDrop. Pay attention to A260/A280 ratio. For highly pure RNA, a ratio of 1.8-2.1 is expected. If necessary, repurify by adding 1/10th nuclease-free 5M NaCl and 1.38 volumes 200 proof pure ethanol before repassing the sample over a fresh mir Vana Filter Cartridge. Continue the total RNA isolation from Step 17.
Assess RNA quality by running the RNA 6000 Nano Assay on an Agilent 2100 Bioanalyzer. A RIN score of >7.0 is normal for total RNA isolated from placenta.
