Tips for RNA Extraction

This week, I wanted to talk about the technique of RNA extraction. Typically, there are two different ways to do this; via RNA extraction kits such as those from Qiagen (Cat No. 74004) or Thermofisher (Cat No. 12183020), or via the phenol-chloroform technique. Both have their advantages and disadvantages; the extraction kits provide a quick and easy protocol, but frequently result in more dilute RNA samples and are a lot more expensive. The phenol-chloroform technique, although a lot more time consuming, produces a higher RNA yield and is a lot more cost effective. As the extraction kits do not offer for a lot of protocol manipulation, today we shall focus on how to best optimise your phenol-chloroform RNA extraction to ensure that you get the highest and purest amount of RNA you can from your sample.

To make it easier to dissect the different steps of the RNA isolation protocol, let’s quickly go through it. First, you lyse your cells/ tissue with an RNA extraction solution like RNAzol/ TRIzol. You next add chloroform to the solution and spin it down to ensure phase separation and aliquot the clear bit of sample (containing your RNA) into a fresh Eppendorf tube. Next you add isopropanol to the solution and spin it down to precipitate the RNA. Afterwards, you discard the supernatant and wash the RNA pellet with 75% ethanol, air dry it, and resuspend it in H2O. And now, onto the tips.

Ensure absolute cleanliness of your lab bench space:

RNAzes, or enzymes that degrade RNA, are everywhere. They are on your bench, they are in the air, they are on your hair. That’s why typically before starting RNA extraction I like to ensure that I am working in the most sterile way as possible. The lab bench is sprayed with either 75% ethanol or a solution called RNAze away (Thermofisher Cat. No 7000TS1), hair is tied back, gloves are changed, filtered pipet tips are fetched and a box of Eppendorf tubes is autoclaved.

Add an extra chloroform extraction step:

I’ve found a paper that demonstrated that they were able to increase their RNA purity by adding an extra chloroform step to their RNA extraction protocol (1). To do this, simply transfer the RNA containing aqueous solution from your first phase separation step into a new Eppendorf tube containing a new tube containing 100μL (per 500μL of extraction solution), vortex, let stand at room temperature for around 3 minutes, and centrifuge once again.

Extend the Ethanol wash step if your aqueous RNA solution has become contaminated:

From my experience, there are two crucial steps in RNA extraction that you have to watch out for to make sure you end up with a pure RNA sample. The first step to be careful of, is looking at the colour of your solution during the RNA precipitation step (adding isopropanol to your aqueous solution). If you add the isopropanol and you see that there is a little bit of a white haze in your solution, that might indicate that you accidentally contaminated your RNA with the interphase layer during the separation phase. One step to try and clean it is to extend the ethanol washing step; if I am particularly worried about my sample I’ll leave it in ethanol for 1 hour on ice. Otherwise, instead of leaving it in ethanol, you could increase the number of times you repeat the ethanol washing step. 

Ensure your samples have properly air dried:

The second step where you might decrease the RNA purity is during the air-drying step. After you discard the ethanol, it is important to ensure that the pellet dries properly, or else the left over phenol shall wreck havoc on your RNA purity ratios. When I first started out doing RNA extractions, I always rushed this step because I was worried that either the pellet would get contaminated or dry out too much. However, I cannot stress enough of critical it is to wait until all the ethanol has evaporated. If a pellet is small to medium size, when it air dries it’ll become clear- leaving the tube looking like its completely empty! That’ll be your sign that you are ready to go. If you are worried about contamination, you can leave your sample tubes in the tissue culture hood, the sterile airflow should ensure that nothing goes wrong!

DEPC-Treated Water is your friend:

When dissolving your pellet in water, I’d highly recommend buying a bottle of DEPC-treated water. DEPC destroys the enzymatic activity of RNAses. Resuspending your pellet sample in around 13μL of DEPC H2O typically yields a good RNA concentration for further reverse transcription to cDNA. Finally, don’t forget to store your samples at -80 degrees to prevent degradation!

That’s all for this week, I hope you guys have found these tips useful in your journeys of RNA extraction!

Citation:

1.        Toni LS, Garcia AM, Jeffrey DA, Jiang X, Stauffer BL, Miyamoto SD, et al. Optimization of phenol-chloroform RNA extraction. MethodsX. 2018 Jan 1;5:599–608.

https://www.sciencedirect.com/science/article/pii/S2215016118300773