Sep 21, 2021

Public workspaceNuclear RNA purification V.2

This protocol is a draft, published without a DOI.
  • 1University of Leicester
  • Michael Tellier
Michael Tellier
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Protocol Citation: Michael Tellier 2021. Nuclear RNA purification. protocols.io https://protocols.io/view/nuclear-rna-purification-byfhptj6Version created by Michael Tellier
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 21, 2021
Last Modified: September 21, 2021
Protocol Integer ID: 53449
Keywords: RNA, purification, cellular fractionation, sub-cellular, nucleus,
Abstract
Protocol for purifying nuclear RNA for qRT-PCR or next generation sequencing analysis.
Day 1
Day 1
  1. Split the cells to obtain a ~80% confluent 10 cm dish on Day 2.
Day 2
Day 2
RNA extraction
  1. Wash the cells twice with ice-cold PBS.
  2. Scrap the cells in 1.2 ml of ice-cold PBS.
  3. Centrifuge at 1,000 rpm for 5 minutes at 4°C.
  4. Resuspend the pellet with slow pipetting in 1 ml of Lysis Buffer B (10 mM Tris-HCl pH 8, 140 mM NaCl, 1.5 mM MgCl2, 0.5 % NP-40).
  5. Centrifuge at 1,000 g for 3 minutes at 4°C.
  6. Resuspend the pellet with slow pipetting in 1 ml of Lysis Buffer B and transfer to an ice-cold 14 ml round-bottom tube.
  7. Add drop by drop 100 μl of the Detergent Stock Solution (3.3 % (w/v) sodium deoxycholate, 6.6 % (v/v) Tween 40) under slow vortexing.
  8. Transfer to a fresh ice-cold 1.5 ml tube. Centrifuge at 1,000 g for 3 minutes at 4°C.
  9. Resuspend the pellet with slow pipetting in 1 ml of Lysis Buffer B.
  10. Centrifuge at 1,000 g for 3 minutes at 4°C.
  11. Resuspend the pellet in 1 ml of TRIzol using a 21-gauge syringe and incubated 5 minutes at room temperature.
  12. Add 200 μl of chloroform and vortex the sample vigorously for 15 seconds.
  13. Centrifuge at 12,000 g for 15 minutes at 4°C.
  14. Transfer the aqueous fraction to a new tube containing 580 μl of isopropanol.
  15. Incubate 10 minutes at room temperature.
  16. Centrifuge at 12,000 g for 10 minutes at 4°C.
  17. Remove most of the supernatant with a 1 ml pipette.
  18. Centrifuge at 12,000 g for one minute at 4°C.
  19. Remove the remaining liquid with a 10 or 20 μl tip.
  20. Resuspend the pellet in 87 μl of RNase-free water, 10 μl of 10 × DNase buffer, 2 μl of DNase I, and 1 μl of RNase OUT.
  21. Incubate for 30 minutes at 32°C.
  22. Add 100 μl of acid-phenol:chloroform pH 4.2.
  23. Vortex 10 seconds, then centrifuge 5 minutes at 13,000 rpm at room temperature.
  24. Transfer the upper phase to a new tube and add 100 μl of acid-phenol:chloroform pH 4.2.
  25. Vortex 10 seconds, then centrifuge 5 minutes at 13,000 rpm at room temperature.
  26. Transfer the upper phase to a new tube and add 250 μl of 100% ethanol, 10 μl of NaOAc, and 1 μl of Glycoblue (or 1 μl of glycogen 10 mg/ml).
  27. Invert the tube several times and incubate at -20°C for at least two hours (or overnight).
  28. Centrifuge for 20 minutes at 13,000 rpm at 4°C.
  29. Remove the supernatant and centrifuge for two minutes at 13,000 rpm at 4°C.
  30. Remove the last drops, air dry for 1-2 minutes, and resuspend in 20 ul of RNase-free water.
  31. Determine the concentration and the 260/280 and 260/230 ratios using a NanoDrop or another system.
  32. Perform the cDNA reaction for qRT-PCR or NGS.