Aug 22, 2018
Version 2
RNA Extraction with Trizol for cells V.2
- Maria Cármen Sales1,
- Maryana Branquinho1,
- Maysa Silva1
- 1Universidade de São Paulo
Protocol Citation: Maria Cármen Sales, Maryana Branquinho, Maysa Silva 2018. RNA Extraction with Trizol for cells . protocols.io https://dx.doi.org/10.17504/protocols.io.ssheeb6
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: August 21, 2018
Last Modified: August 22, 2018
Protocol Integer ID: 14889
Keywords: rna extraction with trizol, rna extraction, trizol, rna, extraction, cell
Materials
MATERIALS
RNAse-free Water
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Isopropanol
ChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #319988
0.5% Sodium dodecyl sulfate solution
Ethanol 75%
0.1 mM EDTA - Ethylenediaminetetraacetic acid
Troubleshooting
Safety warnings
The triazole reagent is toxic, gloves, lab coat, mask should be used. Manipulate the trizol in the exhaust hood.
Before start
Clean the work area with 70% alcohol. Use all filters and autoclaved tips. Refrigerate your centrifuge to 4C.
Remove the culture medium from the plate and add 750μl of Trizol to 1 x 105-107 cells.
Homogenize with the pipette and transfer to a tube.
In this step, you can freeze this sample for up to 6 months in the -80C freezer or continue the protocol.
750 µL Trizol
-80 °C freezer
Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex.
00:05:00 Incubation
Add 200 μl L of chloroform per 1 mL of Trizol and incubate for 2 to 3 minutes.
00:02:00 Incubation
Centrifuge the sample for 15 minutes at 12,000 xg at 4°C.
The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase.
4 °C Centrifugation
00:15:00 Centrifugation
Transfer the aqueous (incolor) phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.
Add 500 μl of isopropanol per 1 mL of Trizol. Incubate for 10 minutes.
00:10:00 Incubation
Centrifuge for 10 minutes at 12,000 × g at 4°C.
4 °C Centrifugation
00:10:00 Centrifugation
Discard the supernatant with a micropipetto and resuspend the pellet in 1 mL of 75% ethanol per 1 mL of Trizol.
Vortex the sample briefly and centrifuge for 5 minutes at 7500 × g at 4°C.
4 °C Centrifugation
00:05:00 Centrifugation
Discard the supernatant with a micropipettor. Vacuum or air dry the RNA pellet for 5–10 minutes.
00:05:00 Vacuum or air dry
Resuspend the pellet in 20–50 µL of RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting up and down.
20 µL RNase-free water, 0.1 mM EDTA, or 0.5% SDS solution
Incubate in a water bath or heat block set at 55–60°C for 10–15 minutes.
55 °C water bath
00:10:00 water bath
Store in freezer -80°C until use or make the cDNA reaction then.
-80 °C storage