Sep 23, 2025
RNA Extraction from Cultured Human Dorsal Root Ganglia
- Joesph B Lesnak1,
- Theodore Price1
- 1University of Texas at Dallas
- PRECISION Human Pain Network
Protocol Citation: Joesph B Lesnak, Theodore Price 2025. RNA Extraction from Cultured Human Dorsal Root Ganglia. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1yp9lmk/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: September 16, 2024
Last Modified: September 23, 2025
Protocol Integer ID: 107714
Keywords: rna extraction from cultured human dorsal root ganglia, rna extraction from cultured hdrg neuron, cultured human dorsal root ganglia, cultured hdrg neuron, rna extraction
Funders Acknowledgements:
NIH
Grant ID: U19NS130608
Abstract
This protocol was developed to perform RNA extraction from cultured hDRG neurons using Qiagen
RNeasy Plus Micro kit
Materials
| Material | Company | Product Number | |
| RNAeasy Plus Micro Kit | Qiagen | 74034 | |
| DPBS | Cytiva | SH30264.FS | |
| gDNA Eliminator Spin Column | Qiagen | 1030958 | |
| RLT Plus Buffer | Qiagen | 1030963 | |
| Beta Mercaptoethanol | Fisher Scientific | BP176-100 | |
| 100% Ethanol | Fisher Bioreagents | BP2818-500 | |
| MinElute Spin Columns | Qiagen | 1026497 | |
| RW1 | Qiagen | 1015763 | |
| RNAse Free DNAse Set | Qiagen | 79256 | |
| Reconstituted DNAse | Qiagen | 1010395 | |
| RDD Buffer | Qiagen | 1010397 | |
| RPE | Qiagen | 1017974 | |
| Ultrapure Distilled Water | Invitrogen | 10977-015 | |
| Cell Scraper | Fisher Scientific | 08-100-241 | |
| 1.7mL Microcentrifuge Tube | Costar | 3621 | |
| 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.
Preparation
Cell cultures are prepared as described in dx.doi.org/10.17504/protocols.io.kxygxyd64l8j/v1
Prepare RLT Plus containing Beta mercaptoethanol in separate tube in fume hood
Prepare 350 ul of RLT Plus lysis buffer for each sample
Add Beta mercaptoethanol 1:100 in RLT Plus Buffer
Example: Add 10 ul of Beta mercaptoethanol to 1ml of RLT Plus
Vortex
Prepare 70% ETOH by diluting 100% ETOH in Ultrapure Distilled Water
Prepare 80% ETOH by diluting 100% ETOH in Ultrapure Distilled Water
RNA Extraction
Aspirate media and wash cells 1x with 500ul of 1X DPBS
Performing one well at a time, aspirate out DPBS and add 350ul of RLT plus buffer with Beta mercaptoethanol to each well
Be careful when pipetting as this is soapy and will form a lot of bubbles if not careful
Scrape down cells using cell scraper and collect RLT plus buffer and place in 1.5ml tube
Vortex all samples for 30 seconds
Transfer the lysate to a gDNA eliminator spin column place in a 2ml collection tube
Centrifuge for 30 seconds at ≥8000g at room temperature
Discard column and SAVE flow-through
Add 1 volume of 70% ETOH (usually around 350ul) to the flow-through and mix well by pipetting
Transfer the sample to an RNeasy MinElute spin column placed in 2ml collection tube
Centrifuge for 15 seconds at ≥8000g at room temperature
Discard flow-through
Add 350 ul of RW1 to RNeasy MinElute spin column
Centrifuge for 15 seconds at ≥8000g at room temperature
Discard flow-through
DNAse clean up step
Add 70ul of RDD buffer to 10ul aliquots of reconstituted DNase
Add 80ul of RDD buffer + DNase directly to spin column
Incubate for 15 minutes at room temperature
Add 350 ul of RW1 to RNeasy MinElute spin column
Centrifuge for 15 seconds at ≥8000g at room temperature
Discard flow-through
Add 500 ul of RPE RNeasy MinElute spin column
Centrifuge for 15 seconds at ≥8000g at room temperature
Discard flow-through
Add 500 ul of 80% ETOH to RNeasy MinElute spin column
Centrifuge for 2 minutes at ≥8000g at room temperature
Discard flow-through and collection tube
Place MinElute spin column in new 2ml collection tube
Open lid of spin column
Centrifuge for 5 minutes at full speed (16,000g) at room temperature
Discard flow-through and collection tube
Place MinElute spin column in new 1.5ml collection tube
Place 14ul of DNA/RNA free water directly to center of spin column
Incubate for 1 minute at room temperature
Centrifuge for 1 minute at full speed (16,000g)
Keep collection tube, and discard column
Freeze and store RNA by placing in -80°C freezer