Nov 21, 2024

Public workspaceRNA Extraction using Trizol

DOI
dx.doi.org/10.17504/protocols.io.x54v922pml3e/v1
  • 1Washington University
Cecilia Escudero
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DOI: dx.doi.org/10.17504/protocols.io.x54v922pml3e/v1
Protocol Citation: Carolina Lopez 2024. RNA Extraction using Trizol. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v922pml3e/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: May 30, 2024
Last Modified: November 21, 2024
Protocol Integer ID: 100946
Abstract
Protocol for RNA extraction from cell lines and tissue
Materials

ABC
ReagentSourceIdentifier
TRIzol ReagentLife TechnologiesREF#15596018
Linear AcrylamideAmbionCAT#AM9520

RNA Extraction using Trizol
RNA Extraction using Trizol
1h 15m 15s
1h 15m 15s
From cell lines:
1. Add Amount1 mL of TRIZOL for RNA isolation.  
2. Add Amount200 µL of chloroform and shake by Duration00:00:15 vigorously.
3. Leave for 3-10 min at RT to allow for phase separation.
4*. Centrifuge for Duration00:15:00 at Max speed, Temperature4 °C *
5. Take 3/4 of the upper aqueous phase (very carefully).
6. Mix with the same volume of cold 100% isopropanol as the volume taken from the aqueous phase. (500 μL)
- Optional step:
  ◊ Add Amount7 µL of acrylamide (if you expect too low amount of RNA from the sample, typically don’t need if extracting from 6 well or larger)
7. Gently invert.
8*. Leave for Duration00:10:00 at RT or longer at Temperature-20 °C (If expecting low yield, leave O/N at Temperature-20 °C )
9. Centrifuge for Duration00:15:00 at Max at Temperature4 °C .
10. Discard the supernatant with care.
11. Wash the pellet by Amount1 mL of 75% (or 70%) Cold EtOH.
12. Vortex (or pipette) the sample briefly to completely wash pellet.
13. Centrifuge for Duration00:10:00 at max speed at Temperature4 °C .
14. Remove the supernatant.
  • Remove supernatant by first dumping into waste container, then draining onto paper towel
  • Spin tubes down in microfuge
  • Remove residual EtOH from the pellet.
15. Dry the pellet for about 10-20min.
  • Dry upside down on kimwipe
  • Some people like to dry on the air-intake of the hood
16*. Resuspend the pellet with Amount15 µL of dH2O
17. Incubate at Temperature65 °C for Duration00:05:00 .

*steps different when extracting RNA from in vivo samples
4. Spinning for Duration00:20:00 can help remove “white floaties” from aqueous phase
8. Do not let RNA precipitate for longer than 10 min, usually can start centrifuge step right away
16. Resuspend in Amount50-60 µL of dH2O
1h 15m 15s
From tissue samples:
* Indicate step modification when extracting RNA from tissue:
4. Spinning for Duration00:20:00 can help remove “white floaties” from aqueous phase
8. Do not let RNA precipitate for longer than 10 min, usually can start centrifuge step right away
16. Resuspend in Amount50-60 µL of dH2O