Feb 01, 2020
Version 1
RNA Extraction for RIN and DV 200 Analysis V.1
- Jamie Allen1,
- Elizabeth Neumann1,
- Maya Brewer1,
- Jeff Spraggins1,
- Danielle Gutierrez1,
- Mark De Caestecker2
- 1Vanderbilt University;
- 2Division of Nephrology, Vanderbilt University Medical Center
- VU Biomolecular Multimodal Imaging Center / Spraggins Research Group
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Protocol Citation: Jamie Allen, Elizabeth Neumann, Maya Brewer, Jeff Spraggins, Danielle Gutierrez, Mark De Caestecker 2020. RNA Extraction for RIN and DV 200 Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.86nhzde
Manuscript citation:
Adapted from: Masato Hoshi, MD, PhD, Jain Lab, Division of Nephrology, Washington University School of Medicine
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: November 09, 2019
Last Modified: October 19, 2023
Protocol Integer ID: 29614
Keywords: HuBMAP, Kidney, Quality Assessment, RNA Quality, RIN, DV 200
Abstract
Scope: Extract RNA for RIN and DV 200 Assessment.
Expected Outcome: RIN and DV 200 measurements for tissue quality assessment.
Materials
Materials
1. Forceps
2. 1.5mL microcentrifuge tubes
3. 23G and 27G needle syringes
4. TRIzoILS
5. DEPC treated water
6. RNase free water
7. Phase Lock Gel, 5PRIME #2302830
8. Chloroform
9. Centrifuge
10. Isopropanol
11. Ethanol
12. Qiagen RNeasy Plus Micro Kit
13. 2-ME
14. RPE Buffer
15. Bioanalyzer
Solutions:
1. Glycogen Solution
5 mg/mL
Protocols:
Qiagen RNeasy Plus Micro Kit
Homogenize Tissue
Homogenize Tissue
Place tissue section in 750 μL of TRIzoILS.
Homogenize the sample:
Use a homogenizer or pass sample through a 23G then a 27G needle syringe.
Freeze samples on dry ice and re-thaw once. Mix well by vortexing.
Add RNase free water to a final volume of 960 μL.
Add 40 μL of glycogen solution (5mg/mL, making a final conc of 200 μg/mL of glycogen) to each tube and mix well.
Loading
Loading
Incubate for 5 minutes at 20oC (room temperature).
Add 250 μL of chloroform and shake for 15 seconds.
Centrifuge at 12,000 x g for 10 minutes at 4oC.
Move supernatant to a new 1.5 mL tube.
Add 600 μL isopropanol.
Vortex and incubate the tube at -20oC for 20 minutes.
Centrifuge at 20,000 x g for 20 minutes at 4oC.
Discard supernatant and add 600 μL 80% Ethanol.
Vortex and centrifuge at 20,000 x g for 5 minutes at 4oC
Discard supernatant and air dry for a few minutes.
Add 300 μLof RLT plus buffer (from Qiagen RNeasy Plus Micro Kit) containing 2-ME (10 μL of 2-ME for 1 mL of RLT Plus Buffer, 1% 2-ME) and mix.
Add 450 μL 100% ethanol (1.5 volumes) and mix well.
Transfer 750 μLof the sample to RNeasy MinElute spin column and centrifuge for 1 min at 12,000 x g at 24oC.
Collection
Collection
Centrifuge Phase Lock Gel to move gel to the bottom of column (12,000 x g for 5 minutes).
Then transfer the TRIzol mixture mixture to the column.
Add 500 μL of RPE buffer to the column and centrifuge at 12,000 x g for 1 min at 24oC.
Discard flow through and add 500 μL RPE buffer to the column. Centrifuge at 12,000 x g for 2 min at 24oC.
Transfer column to a new collection tube. Discard the old collection tube with the flow through.
Transfer the collected flow through back through the same column and centrifuge for 1 min at 12,000 x g at 24oC. (This is a double application of sample). This time discard the flow.
Open the lid of the column and centrifuge at 12,000 x g for 5 min at 24oC to dry up the column membrane completely.
Place the column in a new 1.5 mL tube and add 10 uL of RNase free water on the center of the column membrane. Incubate for 2 min at 20oC.
Centrifuge at 12,000 x g for 3 min at 24oC.
Add 10 uL of RNase free water again on the center of the column membrane and incubate for 2 min at 20oC.
Centrifuge at 12,000 x g for 3 min at 24oC (this is a double elution).
Total volume should be ~20 uL.
Provide VANTAGE Core with 10 uL of solution for RIN and DV 200 analysis.