Dec 02, 2024

Public workspaceBulk RNAseq extraction

DOI
dx.doi.org/10.17504/protocols.io.bp2l6d1okvqe/v1
  • Kimberly Paquette1,
  • Andrew Pyman2,
  • Jose Bras3
  • 1Center for Alzheimer's and Related Dementias, National Institute on Aging, Bethesda, Maryland, USA;
  • 2Van Andel Institute, Grand Rapids, Michigan, USA;
  • 3Regeneron Pharmaceuticals, Tarrytown, New York, USA
Kimberly Paquette
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DOI: dx.doi.org/10.17504/protocols.io.bp2l6d1okvqe/v1
Protocol Citation: Kimberly Paquette, Andrew Pyman, Jose Bras 2024. Bulk RNAseq extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6d1okvqe/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: December 02, 2024
Last Modified: December 02, 2024
Protocol Integer ID: 113448
Keywords: ASAPCRN, RNA , Brain, Extraction
Funders Acknowledgements:
ASAP
Grant ID: ASAP-024407
Abstract
RNA extraction from frozen brain using Qiagen's RNeasy Lipid Tissue Mini kit.
Attachments
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Materials



ReagentManufacturerCat no.
RNeasy Lipid Tissue Mini Qiagen74804
ChloroformMillipore SigmaC2432
Pure EthanolMillipore SigmaE7023
Qubit RNA Broad Range kitThermo Fisher Scientific Q10210
TissueRuptor II Qiagen9002755
TissueRuptor II probesQiagen990890
RNase ZapThermo Fisher Scientific AM9780
RNase-Free DNase setQiagen79254

Cutting Brain Tissue
Cutting Brain Tissue
Manually cut 40-50mg of frozen brain tissue on dry ice. Weigh and place back in Temperature-80 °C freezer until ready for extraction.

Note: do not let tissue thaw. Keep frozen at all times.

RNA extraction
RNA extraction
Using the Qiagen RNeasy Lipid Tissue Mini kit, prepare reagents and materials for extraction in a chemical hood.

Note: use an RNase free environment and clean with RNase Zap.
Toxic
Follow extraction steps outlined in Qiagen RNeasy Lipid Tissue Mini kit using the Tissue Ruptor II to homogenize frozen brain tissue.
Include on column DNase digestion step.

Note: keep extracted RNA on wet ice.
Quality Control
Quality Control
Using the Qubit RNA Broad Range assay kit, measure the concentration of extracted RNA using Amount2 µL of sample.

Aliquot desired amount of sample to continue with for sequencing (Amount>500 ng ) .

Optional: sequence with Illumina's TruSeq stranded riboreduced total RNA at the recommended 50M reads/sample.
Storing RNA
Storing RNA
Snap freeze each RNA sample with liquid nitrogen and store in Temperature-80 °C freezer indefinitely.