Jan 21, 2025
RNA Isolation from Cultured Mammalian Cells Using TRIzol Reagent
- Anand Kumar Veeramachineni1
- 1Springer Nature
Protocol Citation: Anand Kumar Veeramachineni 2025. RNA Isolation from Cultured Mammalian Cells Using TRIzol Reagent. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo98b7v4o/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: January 13, 2025
Last Modified: January 21, 2025
Protocol Integer ID: 118188
Abstract
This protocol is intended to isolate total RNA from cultured mammalian cells using TRIzol reagent, facilitating downstream applications such as RT-PCR, Northern blotting, and gene expression analysis.
Materials
Materials -
TRIzol Reagent 5 mL
Chloroform 1.5 mL
Isopropanol 1.2 mL
Ethanol (75%) 1.5 mL
RNAse-free Water 1 mL
Microcentrifuge tubes 5 tubes
Pipette tips 5 tips
Cell scraper 1
Ice As needed
Reagents -
TRIzol Reagent
Chloroform
Isopropanol
Ethanol (75%)
Safety warnings
Always wear gloves, lab coat, and safety goggles when handling TRIzol, chloroform, and isopropanol. Work in a fume hood when using volatile organic solvents.
Cell Lysis and RNA Extraction
Cell Lysis and RNA Extraction
Remove the culture medium from the cells using a vacuum or pipette. Add 1 mL of cold TRIzol reagent to the cell culture dish. Scrape the cells into the TRIzol using a sterile cell scraper. Transfer the cell suspension to a 1.5 mL microcentrifuge tube on ice. Vortex the tube for 10 seconds to ensure complete lysis.
Add 0.2 mL of chloroform to the tube. Vortex for 15 seconds and let it sit at room temperature for 2-3 minutes. Centrifuge the tube at 12,000 g for 15 minutes at 4 °C.
RNA Precipitation
RNA Precipitation
Recover the upper aqueous phase (approximately 0.5 mL) into a new microcentrifuge tube. Add 1.25 mL of isopropanol to the aqueous phase. Invert the tube several times to mix. Incubate the tube at -20 °C for at least 1 hour to precipitate RNA.
Centrifuge the tube at 12,000 g for 30 minutes at 4 °C to pellet the RNA. Discard the supernatant and wash the RNA pellet with 1 mL of 75% ethanol. Centrifuge again at 7,500 g for 5 minutes at 4 °C.
Protocol references
https://experiments.springernature.com/articles/10.1385/0-89603-491-7:315
https://experiments.springernature.com/articles/10.1038/nprot.2006.83
https://experiments.springernature.com/articles/10.1385/1-59259-038-1:47
https://experiments.springernature.com/articles/10.1385/1-59259-850-1:139
https://experiments.springernature.com/articles/10.1038/nprot.2006.143