Sep 16, 2025
Total RNA isolation from cells using RNAqueous-Micro Total RNA Isolation Kit
- Peng Xu1,2,
- Pietro De Camilli1,2
- 1Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Peng Xu, Pietro De Camilli 2025. Total RNA isolation from cells using RNAqueous-Micro Total RNA Isolation Kit . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg318zzl25/v1
Manuscript citation:
Defect in hematopoiesis and embryonic lethality at midgestation of Vps13a/Vps13c double knockout mice
Peng Xu, Rubia Isler Mancuso, Marianna Leonzino, Caroline J. Zeiss, Diane S. Krause, Pietro De Camilli
bioRxiv 2025.05.09.653147; doi: https://doi.org/10.1101/2025.05.09.653147
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, 2025
Last Modified: September 16, 2025
Protocol Integer ID: 227444
Keywords: micro total rna isolation kit, isolation of total rna, total rna isolation, total rna, small number of cell, isolation
Funders Acknowledgements:
National Institutes of Health
Grant ID: NS36251
National Institutes of Health
Grant ID: DA018343
Aligning Science Across Parkinson
Grant ID: ASAP-000580
Yale Cooperative Center of Excellence in Hematology
Grant ID: U54DK106857
Abstract
For the isolation of total RNA from a small number of cells
Troubleshooting
Sample disruption
Resuspend cell pellet (up to ~500,000 cells) by vortexing vigorously in at least 100 μL Lysis Solution.
RNA isolation
For a standard prep of 100 μL of lysate, add 50 μL of 100% ethanol, and vortex briefly but thoroughly.
Load the lysate/ethanol mixture (up to 150 μL) onto a Micro Filter Cartridge Assembly and close the cap.
Centrifuge for ~10 sec at maximum speed or until all of the mixture has passed through the filter. Longer centrifugation times may be needed to filter the lysate from tissue samples. For lysate/ethanol mixtures >150 μL, load and filter the first 150 μL, then repeat with additional aliquots until the entire sample has passed through the filter. The Collection Tube has a capacity of ~700 μL when assembled with a Micro Filter Cartridge; if more than 150 μL of lysate/ethanol mixture is filtered, empty the Collection Tube before proceeding. The RNA is now bound to the filter in the Micro Filter Cartridge.
Open the Micro Filter Cartridge, add 180 μL of Wash Solution 1 (working solution mixed with ethanol) to the filter and close the cap. Centrifuge for ~10 sec to pass the solution through the filter.
Open the Micro Filter Cartridge, add 180 μL of Wash Solution 2/3 (working solution mixed with ethanol) to the filter and close the cap. Centrifuge for ~10 sec to pass the solution through the filter. Repeat with a second 180 μL aliquot of Wash Solution 2/3.
Open the Micro Filter Cartridge assembly, remove the filter cartridge from the Collection Tube, and pour out the flow-through. Replace the Micro Filter Cartridge into the same Collection Tube, close the cap, and centrifuge at maximum speed for 1 min to remove residual fluid and dry the filter.
Label a Micro Elution Tube (1.5 mL tubes provided with the kit) and transfer the Micro Filter Cartridge into it.
Apply 5–10 μL of Elution Solution, preheated to 75°C, to the center of the filter. Close the cap and store the assembly for 1 min at room temperature. Centrifuge the assembly for ~30 sec to elute the RNA from the filter.
Repeat with a second 5–10 μL aliquot of preheated Elution Solution,
collecting the eluate in the same Micro Elution Tube.
Protocol references