Sep 23, 2025
Muscle Tissue RNA Extraction
- Joesph B Lesnak1,
- Theodore Price1
- 1University of Texas at Dallas
- PRECISION Human Pain Network
Protocol Citation: Joesph B Lesnak, Theodore Price 2025. Muscle Tissue RNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zxo1gqe/v1
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: August 21, 2025
Last Modified: September 23, 2025
Protocol Integer ID: 225196
Keywords: rna extraction from human lumbar muscle tissue, muscle tissue rna extraction, human lumbar muscle tissue, rna extraction
Funders Acknowledgements:
NIH
Grant ID: U19NS130608
Abstract
A protocol for RNA extraction from human lumbar muscle tissue.
Guidelines
Collection of tissue for this protocol requires prior approval by the user's Institutional Ethics Board or equivalent ethics committee.
Materials
| A | B | C | |
| Material | Company | Product Number | |
| CP02 cyroPREP Automated Dry Pulverizer | Corvaris | 500001 | |
| tissueTUBE TT05M XT Extra Thick | Corvaris | 520140 | |
| 1.7mL Microcentrifuge Tube | Costar | 3621 | |
| Ultrapure Distilled Water | Invitrogen | 10977-015 | |
| RNAzap | Invitrogen | AM9780 | |
| Kimwipes | Kimtech | 06-666A | |
| Buffer RLT | Qiagen | 1015750 | |
| β-mercaptoethanol (BME) | Fisher Scientific | BP176-100 | |
| Proteinase K | Qiagen | 19131 | |
| 100% Ethanol | Fisher Bioreagents | BP2818-500 | |
| RNeasy Spin Column | Qiagen | 1112543 | |
| Buffer RW1 | Qiagen | 1015763 | |
| RNAse Free DNAse Set | Qiagen | 79256 | |
| Reconstituted DNAse | Qiagen | 1010395 | |
| RDD Buffer | Qiagen | 1010397 | |
| Buffer RPE | Qiagen | 1017974 | |
| 2.0mL Collection Tube | Qiagen | 1016810 |
Troubleshooting
Safety warnings
Safety training courses on Bloodborne Pathogens, Biological Safety, Chemical Hygiene, Compressed Gases, Biological Waste Management, Personal Protective Equipment, Autoclave and Media Kitchens, General Laboratory Safety, and Cryogen Safety are required by UTD and can be taken through its BioRAFT system.
Safety Training related to Biomedical Research with Human Subjects is required by UTD and can be taken through its CITI program.
Ethics statement
All human tissue recovery, research and data handling is performed in accordance with the University of Texas at Dallas Institutional Review Board (IRB) regulations.
Prep Work
Remove tissue, cut off a section, and place in a 1.5 ml tube on dry ice. Measure the weight of each tissue and write down
Roughly 30-50 mg of tissue
Try to avoid really blood sections
Can be stored in tubes long term till ready to extract
Prepare Lysis buffer
300 ul needed per sample
Add 10 ul of β-mercaptoethanol (BME) per 1 mL of Buffer RLT
Turn on heat block to 55°C
Extraction RNA
Place tissue pieces in Corvaris TT1 bag and leave on dry ice till ready to pulverize
Dip bag into liquid nitrogen and impact with Corvaris till powder is formed
Pulverize all samples before continuing
Add 300 uL of Buffer RLT + BME lysis buffer to bag to retrieve frozen, pulverized samples, and place back in 1.5 mL tube
Mix up and down with pipette tip to get all powdered sample around bag
Add 590 uL of DNA/RNAse free water to each sample
Add 10 uL of proteinase K to each sample
Mix with hand by turning over and place in heat block at 55°C for 15 minutes
Centrifuge sample for 10,000g at room temp for 3 minutes
Transfer supernatant to new tube
Add 0.5 volume of 100% ETOH (~450 ul) to each sample and mix by hand, do not vortex
Load 700 uL of sample onto RNeasy spin column
Centrifuge for 15 seconds >8,000g at room temperature.
Discard the flow through.
Repeat loading until the whole sample has gone through the column.
Add 350 uL Buffer RW1 to the RNeasy spin column.
Centrifuge for 15 seconds >8,000g at room temperature.
Discard the flow through.
Perform DNAase clean up step
Add 70 uL of Buffer RDD (fridge) to aliquots of Qiagen DNAse (10 uL) (-20°C)
Add the total volume from tube (~80 uL) directly on to column
Incubate at room temperature for 15 minutes
Add 350 uL Buffer RW1 to the RNeasy spin column.
Centrifuge for 15 seconds >8,000g at room temperature.
Discard the flow through.
Add 500 uL Buffer RPE onto RNeasy spin column
Centrifuge for 15 seconds >8,000g at room temperature.
Discard the flow through.
Pipette 500 uL Buffer RPE onto the RNeasy spin column.
Centrifuge 2 minutes > 8,000g at room temperature.
Discard the flow through.
Transfer RNeasy spin column to new 2 mL collection tube
Centrifuge RNeasy spin column for 1 minute to dry at full speed (16,000g) at room temperature.
Transfer the RNeasy column to new 1.5 mL collection tube
Pipette 40 uL of RNase-free water; incubate 1 min.
Centrifuge RNeasy column for 15 seconds > 8,000g at room temperature
Measure concentration and A260/A280 and A260/A230 ratios on nanodrop
Store RNA in -80°C