Jan 28, 2022
Total RNA extraction from frozen placenta tissue
- 1University of California, San Diego
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
Protocol Citation: Scott Lindsay-Hewett 2022. Total RNA extraction from frozen placenta tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.b4b9qsr6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 26, 2022
Last Modified: January 28, 2022
Protocol Integer ID: 57441
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 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.
Materials
Equipment
Centrifuge
NAME
Benchtop Centrifuge
TYPE
Eppendorf
BRAND
5405000441
SKU
LINK
Any benchtop centrifuge will suffice
SPECIFICATIONS
Equipment
Block heater
NAME
--
BRAND
--
SKU
Equipment
Vortex mixer
NAME
Any
BRAND
xx
SKU
Equipment
Mini-Beadbeater-16
NAME
high-energy cell disrupter
TYPE
BioSpec
BRAND
607
SKU
LINK
1 speed
SPECIFICATIONS
Equipment
Fume hood
NAME
Fume hood
TYPE
Generic
BRAND
Unknown
SKU
Equipment
Set of micropipettes with rack: 100-1000 µl, 20-200 µl, 2-20 µl, and 0.5-10 µl
NAME
Pipettor set
TYPE
Pipetman
BRAND
QP-1001-07
SKU
LINK
Can use equivalent Pipettors
SPECIFICATIONS
Equipment
Bioanalyzer
NAME
Bioanalyzer
TYPE
Agilent
BRAND
G2991AA
SKU
LINK
Any bioanalyzer will suffice.
SPECIFICATIONS
mirVanaTM miRNA isolation kit
Ethanol Pure 200 proof for molecular biologySigma AldrichCatalog #E7023-500mL
Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)Thermo FisherCatalog #AM9720
Dry IceContributed by users
DNA LoBind Tube 1.5ml EppendorfCatalog #022431021
Nuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932
Filter TipsContributed by users
Bioanalyzer RNA 6000 Nano KitAgilent TechnologiesCatalog #5067
Preparation
Preparation
15m
15m
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 mirVana wash buffers as instructed.
Pull frozen placenta samples (~150-200 mg each) from -80 °C and place on dry ice until ready to process.
Note
Integrity of isolated total RNA will be greater if tissue was preserved in RNAlater as soon as possible after harvesting.
Typically, tissue is submerged in RNAlater overnight. The following day, the RNAlater is removed and the sample moved to -80 °C storage.
Cell lysis and tissue disruption
Cell lysis and tissue disruption
20m
20m
Add approximately 1 mL of 1.0mm zirconia/silica beads to each frozen placenta sample, and 700 µL mirVana Lysis/Binding Buffer.
Note
After addition of beads and lysis buffer, tubes should be almost full. Exclude as much air as possible to reduce foaming.
Safety information
Samples should be in screw-cap microcentrifuge tubes with integral O-rings in the caps. Snap-cap tubes should not be used, unless secured with an adapter.
Load samples immediately into Mini-BeadBeater-16 vial holder ring. Up to 16 samples can be accommodated.
Safety information
IMPORTANT: Rotate the vial holder ring to a position where the small hole in the vial holder ring engages the anti-rotation pin sticking out of the wiggle mechanism. Slide the vial holder down the pin and seat it flat on the wiggle mechanism. Slide the large, black plastic hold-down cap over the stainless steel center bolt, aligning it so that it too slides down the anti-rotation pin. The hold-down cap must make contact with the top of the aluminum wiggle mechanism - not just the tops of the microcentrifuge tubes. Finally, screw on and hand-tighten the black knob firmly. Repeat: Tighten firmly.
Switch on the Mini-BeadBeater-16 and run for 00:02:00 .
2m
Place samples immediately into pre-chilled microcentrifuge and spin for 00:05:00 at maximum speed .
5m
Remove 500 µL lysate, being careful not to draw up particulates, and dispense into a fresh labeled microcentrifuge tube.
Organic extraction
Organic extraction
40m
40m
Add 50 µL mirVana miRNA Homogenate Additive (1:10 volume of original lysate) and vortex to mix. Incubate 00:10:00 On ice .
10m
Add 500 µL Acid-Phenol:Chloroform (1:1 volume of original lysate) and vortex 00:01:00 to homogenize sample.
Note
Acid-Phenol:Chloroform may appear as a clear, homogeneous phenol phase (lower), overlayed by a small aqueous phase (upper). Pipette from the lower, not the upper, phase.
Safety information
Phenol is very corrosive and will severely burn the skin. Safety precautions such as gloves, protective eyewear, a lab coat, and working in a fume hood are critical. Discard contaminated pipette tips in appropriate waste container.
1m
Spin 00:05:00 maximum speed in pre-chilled microcentrifuge.
5m
Carefully remove 350 µL of the top aqueous phase and transfer to a fresh labeled microcentrifuge tube.
Note
It is possible to remove a greater volume to maximize RNA yield, but make sure not to disturb the interphase or organic phase.
Safety information
Discard phenol liquid waste and contaminated tubes in appropriate waste containers.
Total RNA isolation
Total RNA isolation
30m
30m
Add 437.5 µL (1.25 volumes) 200 proof pure ethanol and mix thoroughly.
Transfer up to 700 µL lysate/ethanol mixture to mirVana Filter Cartridge (placed into mirVana Collection Tube) and spin 10000 x g, 4°C, 00:00:15 in pre-chilled microcentrifuge. Discard flow-through. Repeat with remaining volume of lysate/ethanol mixture.
Note
mirVana Filter Cartridges can accommodate up to 700 µL volume - do not overfill. To avoid filter damage, do not spin at speeds greater than 10000 x g .
15s
Add 700 µL mirVana Wash Solution 1 and spin 10000 x g, 4°C, 00:00:15 in pre-chilled microcentrifuge. Discard flow-through.
Note
Tip: flow-through can be aspirated into an appropriately-labeled waste container using a vacuum.
15s
Add 500 µL mirVana Wash Solution 2/3 and spin 10000 x g, 4°C, 00:00:15 in pre-chilled microcentrifuge. Discard flow-through. Repeat wash step.
15s
After discarding flow-through from the last step, spin 10000 x g, 4°C, 00:02:00 in pre-chilled microcentrifuge to remove residual Wash Solution.
2m
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 00:01:00 and then spin 00:00:30 maximum speed in pre-chilled centrifuge to elute RNA.
1m 30s
Transfer eluate from the mirVana Collection Tube to a low bind microcentrifuge tube and store at -80 °C .
Note
Minimize freeze/thaw cycles.
Tip: Store a small aliquot in a separate tube for QC.
Quality control
Quality control
1h 30m
1h 30m
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.
Note
RNA can also be quantitated using Qubit RNA Broad Range Assay.
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.