Oct 23, 2024
Fresh Cell Isolation RNA Extraction
- Malu G Tansey1
- 1University of Florida
Protocol Citation: Malu G Tansey 2024. Fresh Cell Isolation RNA Extraction . protocols.io https://dx.doi.org/10.17504/protocols.io.261gerpd7l47/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: October 23, 2024
Last Modified: October 23, 2024
Protocol Integer ID: 110754
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020527
Abstract
This protocol describes the process of extracting RNA from freshly isolated immune cell populations using the Qiagen RNeasy Mini Kit.
Guidelines
Always wear gloves and spray with RNaseZap and spread solution all over gloves.
Make sure to use RNA only pipettes and filter tips.
Perform all steps of the procedure at room temperature. During the procedure, work quickly.
Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure that the centrifuge does not
cool below 20°C.
Materials
RNeasy mini kit (74106, Qiagen)
QIAshredder (79656, Qiagen)
96-100% Ethanol
Nuclease Free Water (AM9938,Thermo Scientific)
2-mercaptoethanol (M6250, Sigma Aldrich)
RNaseZAP(AM9780, Ambion)
1.5ml tubes
Safety warnings
Wear appropriate PPE and work with concentrated β-ME and ethanol in a chemical safety hood.
Before start
Prepare BME lysis buffer
Add 20 μl β-ME per 1 ml Buffer RLT. Dispense in a fume hood. Buffer RLT containing β-ME can be stored at room temperature for up to 1 month.
Prepare RPE buffer
Add 4 volumes of ethanol (96–100%) as indicated on the bottle to concentrate buffer RPE to obtain a working Solution.
RNA Extraction
RNA Extraction
Resuspend cell pellet in 350uL of BME+RLT lysis buffer and vortex well.
Transfer lysis solution to (purple) qiashredder tubes and then spin 21300xg (full speed) for 2min.
Can freeze flow-through of cell lysate at this stage and store in -80 degrees celsius for several months
Add 350uL of 70% ethanol to the homogenized lysate, and mix well by pipetting.
Transfer 700 μl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube. Close the lid gently, and centrifuge for 30s at 9,391xg (10,000rpm). Discard the flow-through .
Add 700 μL Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 30s at 9,391xg (10,000 rpm) to wash the spin column membrane. Discard the flow-through.
Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 30s at 9,391xg (10,000rpm) to wash the spin column membrane. Discard the flow-through.
Repeat step 6.
Spin for 2 minutes at full speed to ensure any residual buffers are gone.
Place the RNeasy spin column in a new 1.5 ml collection tube. Add 20μl RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 9,391xg (10,000rpm) to elute
the RNA.
After first spin transfer elution back to the membrane of the RNeasy
spin column and spin again for 1 min at 9,391xg (10,000rpm) (re-elution results in
greater RNA yield). Samples are now ready for downstream assays or should be stored at -80 degrees.