Sep 23, 2025
Disc Tissue RNA Extraction and Qubit Measurement
- Joesph B Lesnak1,
- Theodore Price1
- 1University of Texas at Dallas
- PRECISION Human Pain Network
Protocol Citation: Joesph B Lesnak, Theodore Price 2025. Disc Tissue RNA Extraction and Qubit Measurement. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21qm4g1y/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: 225219
Keywords: rna extraction from human lumbar disc tissue, disc tissue rna extraction, rna extraction, human lumbar disc tissue, qubit measurement
Funders Acknowledgements:
NIH
Grant ID: U19NS130608
Abstract
A protocol for RNA extraction from human lumbar disc 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 TT1 Extra Thick | Corvaris | 520007 | |
| 1.7mL Microcentrifuge Tube | Costar | 3621 | |
| Ultrapure Distilled Water | Invitrogen | 10977-015 | |
| RNAzap | Invitrogen | AM9780 | |
| Kimwipes | Kimtech | 06-666A | |
| Qiazol | Qiagen | 1023537 | |
| Chloroform | Fisher Scientific | C298-500 | |
| 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 | |
| Qubit RNA HS Assay Kit | Invitrogen | Q32855 | |
| Qubit RNA HS Reagent (200X) | Invitrogen | Q32855 A | |
| Qubit RNA HS Buffer | Invitrogen | Q32855 B | |
| Qubit RNA HS Standard (0ng/uL) | Invitrogen | Q32855 C | |
| Qubit RNA HS Standard (10ng/uL) | Invitrogen | Q32855 D | |
| Qubit Assay Tubes | Invitrogen | Q32856 |
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.
Store Tissue in RNAlater
Isolate nucleus pulposus (NP) and annulus fibrosus (AF) tissue and freeze immediately in powdered dry ice or place in 5ml tube with 4ml of RNAlater
If in RNAlater place in 4°C refrigerator overnight
Next day remove tissue from RNAlater and freeze in powdered dry ice
Place in new tube and store in -80°C
Extract RNA
Remove tissue, cut off a section, and place in a 1.5 mL tube on dry ice. Measure the weight of each tissue.
Less than 100mg, the smaller the better, especially for AF samples
Prechill tubes to try and keep tissue as frozen as possible
Place tissue pieces in Corvaris TT2 bag and leave on dry ice till ready to pulverize
Dip bag into liquid nitrogen and impact with Corvaris until powder is formed
It is important to move frozen debris around after each impact to thoroughly pulverize
Add 1 mL Qiazol to bag to retrieve frozen, pulverized samples, and place back in 1.5ml tube
Add 200 uL of chloroform to each tube
Shake for 15 seconds and let incubate for 2 minutes at room temp.
Centrifuge at 4°C 12,000 g, 10 minutes
Collect upper aqueous phase. Transfer it to a new microfuge tube.
Add again 200 uL of chloroform to aqueous phase, shake, incubate, and centrifuge.
Collect upper aqueous phase. Transfer it to a new microfuge tube.
Add again 200 uL of chloroform to aqueous phase, shake, incubate, and centrifuge.
Collect upper aqueous phase. Transfer it to a new microfuge tube.
Add same volume of 70% RNAse free ethanol (~300-400 uL).
Mix gently.
Precipitate on ice for 10 minutes.
Load 700 uL of sample onto RNeasy spin column
Centrifuge 15 sec >8,000 g at room temperature.
Discard the flow through.
Repeat until loading until the whole sample has gone through the column.
Add 350 uL Buffer RW1 to the RNeasy spin column.
Centrifuge 15sec >8,000 g at room temperature.
Discard the flow through and collection tube.
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 15sec >8,000 g at room temperature.
Discard the flow through and collection tube.
Perform DNAase clean up step a second time
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 15sec >8,000 g at room temperature.
Discard the flow through and collection tube.
Pipette 500 uL Buffer RPE onto the RNeasy spin column.
Centrifuge 15sec >8,000 g at room temperature.
Discard the flow through.
Pipette 500 uL Buffer RPE onto the RNeasy spin column.
Centrifuge 2 min > 8,000 rpm at room temperature.
Discard the flow through.
Transfer RNeasy column to new 2mL collection tube
Centrifuge for 1 minute to dry collection tube at full speed (16,000g) at room temperature.
Transfer the RNeasy column to 1.5 mL collection tube
Pipette 15 uL RNase-free water; incubate 1 minute.
Centrifuge RNeasy column > 8,000 g, at room temperature for 15 seconds.
Pipette the same 15 uL RNase-free water in collection tube back onto column; incubate 1 minute.
Centrifuge RNeasy column > 8,000 g at room temperature for 1 minute.
Measure RNA concentration with Qubit
Prep working reagent
i. 200 ul per tube
ii. 1 tube for each sample
iii. 2 tubes for standards
iv. 200x stock solution at room temperature
v. Rest of contents should be at 4°C
vi. Need to use on Qubit tubes
Prep standards
i. 10 ul of low standard + 190 ul working reagent
ii. 10 ul of high standard + 190 ul working reagent
Prep samples
i. 1-5 ul of sample + 199-195 ul of working reagent
Vortex and read samples on Qubit machine