TITLE: Total RNA extraction from frozen placenta tissue
AUTHORS: Scott   Lindsay-Hewett

[DESCRIPTION]
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 mirVana miRNA Isolation Kit from Ambion. 

Written steps are adapted from Ambion's manual for the mirVana miRNA Isolation Kit and BioSpec's instructions for the Mini-BeadBeater-16.

[STEPS]
SECTION: Preparation
3. Heat nuclease-free water  to 95 °C.


SECTION: Preparation
4. Pre-cool microcentrifuge to 4 °C.

 

SECTION: Cell lysis and tissue disruption
7. Add approximately 1 mL of 1.0mm zirconia/silica beads to each frozen placenta sample, and 700 µL mirVana Lysis/Binding Buffer.
 


 





SECTION: Cell lysis and tissue disruption
8. Load samples immediately into Mini-BeadBeater-16 vial holder ring. Up to 16 samples can be accommodated.
 

SECTION: Cell lysis and tissue disruption
9. Switch on the Mini-BeadBeater-16 and run for 2 min.

SECTION: Preparation
6. Pull frozen placenta samples (~150-200 mg each) from -80 °C  and place on dry ice until ready to process.
 


SECTION: Preparation
5. Add 200 proof pure ethanol to mirVana wash buffers as instructed.
SECTION: Cell lysis and tissue disruption
10. Place samples immediately into pre-chilled microcentrifuge and spin for 5 min.
SECTION: Cell lysis and tissue disruption
11. Remove 500 µL lysate, being careful not to draw up particulates, and dispense into a fresh labeled microcentrifuge tube.

SECTION: Organic extraction
12. Add 50 µL mirVana miRNA Homogenate Additive (1:10 volume of original lysate) and vortex to mix. Incubate 10 minon ice.

SECTION: Organic extraction
13. Add 500 µL Acid-Phenol:Chloroform (1:1 volume of original lysate) and vortex 1 min to homogenize sample.
 

 

SECTION: Organic extraction
14. Spin 5 min in pre-chilled microcentrifuge.

SECTION: Organic extraction
15. Carefully remove 350 µL of the top aqueous phase and transfer to a fresh labeled microcentrifuge tube.
 

 


SECTION: Total RNA isolation
16. Add 437.5 µL (1.25 volumes) 200 proof pure ethanol and mix thoroughly. 


SECTION: Total RNA isolation
18. Add 700 µL mirVana Wash Solution 1 and spin 10000 x g, 15 s, 4 °C in pre-chilled microcentrifuge. Discard flow-through.

 

SECTION: Total RNA isolation
19. Add 500 µL mirVana Wash Solution 2/3 and spin 10000 x g, 15 s, 4 °C in pre-chilled microcentrifuge. Discard flow-through. Repeat wash step.
SECTION: Total RNA isolation
20. After discarding flow-through from the last step, spin 10000 x g, 2 min, 4 °C in pre-chilled microcentrifuge to remove residual Wash Solution.

SECTION: Total RNA isolation
21. Transfer mirVana Filter Cartridge into a fresh mirVana Collection Tube. Add 40 µL pre-heated nuclease-free water to the center of the filter and close the cap. Incubate 1 min and then spin 30 s in pre-chilled centrifuge to elute RNA.

SECTION: Total RNA isolation
22. Transfer eluate from the mirVana Collection Tube to a low bind microcentrifuge tube and store at -80 °C.
 


SECTION: Quality control
23. 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 mirVana Filter Cartridge. Continue the total RNA isolation from Step 17.
 


SECTION: Total RNA isolation
17. Transfer up to 700 µL lysate/ethanol mixture to mirVana Filter Cartridge (placed into mirVana Collection Tube) and spin 10000 x g, 15 s, 4 °C in pre-chilled microcentrifuge. Discard flow-through. Repeat with remaining volume of lysate/ethanol mixture.


 

SECTION: Quality control
24. 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.
SECTION: Preparation
1. Clean workspace, pipettes, and gloves with RNaseZAP.
SECTION: Preparation
2. Prepare bucket of ice.