A Guide to RNA Extraction

Prep your station and set yourself up for success

Learn the Basic Steps of RNA Extraction

There are several different methods of RNA extraction, but they all involve the same basic steps. Depending on the method, the order of the steps (particularly steps 4-6 in Table 1), will change.

Table 1 Basic Steps of RNA Extraction

Know Your Options—Methods of RNA Extraction

There are several methods of RNA extraction, listed in Table 2, with the most commonly used non-kit-based method being the guanidium-acid-phenol extraction—commonly referred to as TRIzol and TRIreagent (however there are several excellent reagents and commercial kits on the market for the guanidium-acid-phenol extraction method).

Table 2 Common Methods of RNA Extraction

Choosing the right RNA isolation method

Choosing the right method early will save you time and sample, but with the large variety of methods and kits available for RNA extraction, the right method may be difficult to identify.

Answering the following questions will help you select the best method of RNA extraction:

• What is your sample type and how difficult is it to lyse?
• What type of RNA do you need to isolate (e.g., miRNAs, tRNAs, total RNA, etc.)?
• How much RNA does your downstream application require?
• How many samples are you processing—should you consider high-throughput methods?  
• Will certain resuspension solutions interfere with your downstream application?
• Do you need to isolate other molecules from your sample (e.g., proteins or DNA)?

You can obtain high-quality, ready-to-use RNA from virtually any sample with MP Bio’s kits (Table 3), and some of the kits are available for high-throughput needs and have protocols that can be adapted so you can isolate proteins and RNA from a single sample.

Table 3: MP Bio RNA Isolation Kits

Isolating RNA using the guanidium-acid-phenol extraction method

This is a common liquid-liquid extraction method, which may take longer than silica-based columns, but often has a higher recovery of RNA. It works by relying on phase separation by centrifugation of an aqueous sample mixed with a solution of phenol and chloroform. After centrifugation, an upper aqueous layer and a lower phenol layer are clearly formed, with the upper layer containing your target RNA.

Guanidium thiocyanate is a chaotropic agent that is a component of trizol and is important in denaturing proteins that may bind to nucleic acids or degrade RNA.

The pH is incredibly important. Low pH, around 4, ensures that only RNA preferentially remains in the aqueous phase while DNA and protein remain in the organic phenol layer. This differs from DNA extraction, which uses a guanidinium thiocyanate-phenol solution with  pH to 6 or 7, causing DNA and RNA to partition into the aqueous phase.

Isopropyl alcohol can be added to the aqueous phase (after transferred to a fresh tube) to precipitate RNA. The alcohol disrupts the hydration shells neutralizing the negative phosphate backbone of RNA. Instead of water molecules, positively charged metal salt ions neutralize the negatively charged RNA. The neutral RNA begins to aggregate in the aqueous solution, which gets pelleted using centrifugation. Washing steps with 70% ethanol remove salts and DEPC (Diethylpyrocarbonate)-treated water is used to redissolve the RNA pellet.

Why DEPC water? DEPC nonspecifically inhibits RNase by reacting with amine, hydroxy, and thiol groups within enzymes.

General Method of RNA Extraction from Bacterial Cells

1. Pellet bacterial cells that were growing overnight by centrifuging for 2 minutes at 12,000xg 
2. Decant luria broth supernatant
3. Resuspend the pellet in RNA-PLUS™ (comparable to TRIzol) and lyse cells by pipetting several times. Make sure you get the entire pellet—sometimes residual pellet can linger at the bottom of your tube, which will reduce your yield. 
4. Centrifuge for 10 minutes at 4°C at 12,000xg
5. Transfer the supernatant to a fresh tube (Do not invert the tube, it might leak)
6. Add chloroform, shake vigorously (or vortex), and incubate for 3 minutes at room temperature
7. Centrifuge for 15 minutes at 12,000xg. Now you should see an upper, colorless aqueous phase (this contains your RNA) and a lower reddish phenol phase (this contains contaminating proteins).
8. Transfer the upper aqueous phase to a clean RNase-free tube, careful not to transfer any phenol phase
9. Add 100% isopropyl alcohol and incubate for 10 minutes at room temperature to precipitate the RNA
10. Centrifuge at 12,000g for 10 minutes at 4°C 
11. Discard the supernatant 
12. Wash the pellet with 70%-80% ethanol (invert the tube a few times)
13. Centrifuge at 4°C for 5 minutes at 7500xg
14. Discard the ethanol 
15. Air dry the pellet for roughly 10 minutes at room temperature
16. Resuspend the pellet in DEPC-treated water (or your choice of buffer)

Quantitate your RNA and assess the quality

Before using your RNA, you need to know how much you have and that it’s worth using. The most common method for quantitating RNA is UV spectroscopy.

Quantity: A₂₆₀ of 1.0 is roughly 40 µg/mL single-stranded RNA (assuming the pH is ~7.5)

Quality: A₂₆₀/A₂₈₀ of 1.8-2.1 indicates highly purified RNA

Keep in mind that this method will not differentiate between RNA and DNA, so you should consider treating your samples with RNase-free DNase

Store your purified RNA

You’ve successfully isolated RNA! Now you just need to store it to keep it safe and ready to use for your next experiment.

We recommend storing RNA at –80°C in single-use aliquots to avoid using damaged RNA from freeze-thaw cycles and to help prevent RNase contamination.