Whereas DNA can survive for millennia, RNA is short-lived, which is a bummer if you are trying to isolate it. The reason – as we discussed recently – is that RNA is prone to degradation by enzymes called RNases.
Therefore, isolation of total RNA from cells and tissues requires a method that will efficiently isolate the RNA from the samples while also minimizing RNA degradation.
What is total RNA?
But before we continue – let’s define what total RNA is anyway. As you might expect, it is all the RNA molecules found inside a cell. This includes:
mRNA: long protein-coding messenger RNA transcripts, which serve as the instantaneous readout of cellular gene expression under particular conditions
microRNA (miRNA): and myriad of other smaller noncoding RNA molecules, many of which are involved in regulating and silencing gene expression.
Obtaining high-quality intact total RNA is often the first step in many biological applications aimed at measuring gene expression, including RT-qPCT, microarray analysis and RNA-sequencing.
Isolating total RNA, minus the RNases using Trizol
Fortunately, there are ways to inactivate RNases and avoid this sticky mess. As well as the methods mentioned in Nick’s earlier article here in the Nucleic Acid Purification channel Guanidinium thiocyanate-phenol-chloroform extraction is commonly employed. This uses a pink colored chemical solution called TRIzol from Invitorgen, which was first applied for RNA extraction by Piotr Chomczynski and Nicoletta Sacchi in 1987.
TRIzol is a powerful protein denaturant that breaks down protein cell components and inactivates all enzymes, including RNases. TRIzol extraction typically uses acidic phenol-chloroform to confine total RNA in a clear aqueous phase while proteins and cell debris end up in the pink organic layer. RNA can be recovered by precipitation with ethanol, washed and then redissolved in water.
Basic steps in isolating total RNA from cells and tissues using TRIzol:
1a. If isolating RNA from tissues, you will need to homogenize the sample first in 1 ml of TRIZOL reagent per 50 to 100 mg of tissue using a homogenizer. The sample volume should not exceed 10% of the TRIzol volume.
1b. If isolating RNA from adherent cells grown in culture, rinse the cells with ice cold PBS and lyse cells directly in a culture dish or flask by adding 1 ml of TRIZOL Reagent per 10 cm2 area and scraping with cell scraper or pipette tip. Pass the cell lysate several times through a pipette and vortex thoroughly. If your culture is composed of suspension cells, spin cells down to remove old media, wash in PBS lyse cells with 1 ml TRIZOL for up to 10 million cells by pipetting up and down several times.
2. Incubate the homogenized sample for 5 minutes at room temperature to dissociate nucleoprotein complexes, then centrifuge to remove cell debris.
3. Add 0.2 volume of chloroform per 1 volume of TRIZOL Reagent, cap the tubes securely and vortex samples vigorously for 15 seconds.
4. Incubate samples at room temperature for 5 minutes.
5. Centrifuge the samples at no more than 12,000 x g for 15 minutes at 4C. The mixture will separate into lower organic, interphase, and upper aqueous phases that contains RNA.
6. Carefully transfer the upper aqueous phase without disturbing the interphase into fresh tube. The volume of the aqueous phase is usually about 60% of the TRIzol volume used in step 1.
7. Use 0.5 ml of ethanol per 1 ml of TRIZOL to precipitate the RNA from the aqueous phase.
8. Incubate samples at room temperature for 10 minutes and centrifuge at not more than 12,000 x g for 10 minutes at 4C. The RNA precipitate will form a pellet on the side or bottom of the tube, which could be hard to see by eye.
RNA wash and resuspension
9. Remove the supernatant and wash the RNA pellet once with 75% ethanol.
10. Mix the samples by vortexing and centrifuge at no more than 8000 x g for 5 minutes at 4C. Repeat above washing procedure and remove all leftover ethanol.
11. Air-dry or vacuum dry RNA pellet for 5-10 minutes but don’t heat or centrifuge under vacuum. Don’t overdry RNA or it will be hard to redissolve the pelelt.
12. Dissolve RNA in DEPC-treated water by passing solution a few times through a pipette tip.
Once you have your sample redissolved, determine sample concentration and purity by taking OD measurements at 260 nm and 280 nm. The A260/A280 ratio should be above 1.6.
There are many commercially available kits that use columns that trap the RNA following the precipitation step to make subsequent washes and elutions easier and faster.