RNA Extraction Without a Kit

Introduction

This protocol describes how to extract RNA from tissues or cells without a kit. The general steps of this protocol include homogenization/lysis of cells or tissues, extraction of RNA, precipitation, and resuspension. This protocol provides two options: using Solution D (see reagent section for recipe) or using an all-in-one acid guanidinium thiocyanate-phenol solution such as TRIzol®. For the precipitation step, two options are also included: using Isopropanol or Lithium Chloride.

This protocol was adapted from Chomczynski P and Sacchi N, 2006 and TRIzol® User Guide from ThermoFisher Scientific.

Figure 1: A diagram of the different steps in RNA extraction.

Before Starting

• RNA is not as stable as DNA and is susceptible to degradation by heat, RNases, and other enzymes.
  Pro-Tips
  Always wear gloves, and whenever possible, keep RNA sample and reagents cold and work quickly to reduce RNA degradation.
  
  Keep work area, equipment, and reagents RNase-free (Use an RNase decontamination solution, such as RNaseZap® or RNase AWAY®, may be used).
• For more tips on working with RNA, read this blog post on RNA extraction without a kit.

Last Update: April 10, 2020

Equipment

• Refrigerated microcentrifuge
  • If you only have a non-refrigerated microcentrifuge, see step 4 of either protocol before you start.
• Homogenizer
• Vortexer
• -20°C freezer
• -80°C freezer

Reagents

• Solution D (for Protocol Option #1 ):
  • 4 M Guanidinium Thiocyanate
  • 25 mM Sodium Citrate, pH 7.0
  • 0.5% (wt/vol) N-laurosylsarcosine (Sarkosyl)
  • 0.1 M 2-mercaptoethanol
• TRIzol® or similar product such as TRI Reagent®, RNAzol®, QIAzol® (for Protocol Option #2)
• Water-saturated Phenol
• 2 M Sodium Acetate pH 4
• Chloroform/Isoamyl alcohol (49:1)
• 75% Ethanol
• RNase-free water or TE solution
• RNase free tubes: microcentrifuge tubes, 4 mL polypropylene tubes
• RNase decontamination solution like RNase AWAY® or RNaseZAP®
• Isopropanol (for precipitation step, Option A)
• 7.5 M Lithium Chloride (for precipitation step, Option B)
• Glycogen (Optional)

Caution
Make sure to read the SDS (Safety Data Sheet) for safety warnings and hazards for these reagents. Work in a well-ventilated space and under a fume hood when working with the volatile reagents in the list above.

Procedure

Option #1 - Solution D Protocol

Before starting this protocol, make sure to prepare solution D (see reagent section above for the recipe). If using TRIzol®, jump down to the Option #2 - TRIzol® Protocol section below.

1. Homogenize or lyse tissues or cells in Solution D.
   • For tissues: use 1 mL of Solution D per 100 mg of cells.
   • For cultured cells: use 1 mL of Solution D per 1 X 10⁷ cells.
2. Allow sample(s) to sit at room temperature for 5 minutes to allow for dissociation of the nucleoprotein complexes.
   • The effectiveness of your RNA isolation will depend on how effective your cell lysis protocol is. While simple homogenization is effective for most mammalian tissues; more hardy tissues such as bone, or bacteria/yeast/plant samples will require additional steps to effectively lyse open the cells.
3. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL tube. Add the following sequentially to 1 mL of lysate:
   • Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion.
   • Add 1 mL water-saturated phenol, mix thoroughly by inversion.
   • Add 0.2 mL of Chloroform/Isoamyl alcohol (49:1) and then shake vigorously by hand for 10 seconds.
4. Incubate sample(s) for 15 minutes on ice and centrifuge the sample(s) for 15 minutes at 12,000 × g at 4°C to separate RNA from the rest of the tissue/cell lysate.
   Pro-Tip
   Having your samples spin at 4°C helps reduce RNA degradation. If you don’t have access to a refrigerated centrifuge, you can carefully bring a centrifuge into a cold room for centrifugation. Once you’re done using the centrifuge, bring this equipment back to room temperature, as prolonged storage in the cold room may damage it.
5. Using a pipettor, transfer the top, aqueous phase to a new RNAse-free tube.
   • The mixture separates into a bottom organic layer, an interphase layer, and a top, aqueous layer. Take care to not disturb or collect the interphase layer with your pipette tip.
   Pro-Tip
   To collect as much of the top aqueous phase without disturbing the interphase layer, consider using a lower volume pipettor like a p200 to collect a majority of the aqueous phase. You may have to collect twice or more from the same tube, but unlike using a p1000 tip it will give you more control of where you’re aiming your tip in the tube.
6. Precipitate your sample(s). You can use either Isopropanol or Lithium Chloride for this step.
   • Isopropanol (Option A): Add 1 volume of Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour.
   • Lithium Chloride (Option B): LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of 7.5 M LiCl solution to bring the concentration of LiCl in the extracted aqueous layer to 2.5 M. Incubate at -20°C for 1 hour.
   Pro-Tips
   If you anticipate your RNA yield to be small, RNase-free Glycogen may be used as a carrier to facilitate RNA precipitation. This does not affect the quality of RNA or downstream applications.
   
   To improve yield of RNA, instead of incubating at -20°C for 1 hour, you can try incubating at -80°C overnight.
7. Centrifuge for 20 minutes at 10,000 x g at 4°C and discard the supernatant. There should be a gel-like white pellet of total RNA in the bottom of the tube.
8. Wash the RNA by resuspending the pellet in 0.5–1 ml of 75% Ethanol and vortex for a few seconds. Centrifuge for 5 minutes at 10,000 x g at 4 °C and remove the supernatant.
9. Remove as much of the ethanol wash as possible without disturbing the pellet. Air-dry the pellet for 5-10 minutes.
   Critical
   It is important to not let the pellet get too dry before resuspending, as this affects the solubility of the RNA.
   Pro-Tip
   To prevent overdrying, watch the pellet and carefully remove any residual ethanol wash and add RNase-free water or TE as soon as the entire tube is dried but while the white pellet is still visible.
10. Resuspend RNA pellet in RNase-free water or TE. Quantify and assess the quality of your RNA sample(s) using a spectrophotometer (such as a Nanodrop), agarose gel, or bioanalyzer. For more information on nucleic acid quantification, see our protocol for DNA quantification, which can be modified for RNA. Store your RNA sample(s) at -80 °C to prevent RNA degradation and avoid multiple freeze-thaw cycles.
    Pro-Tip
    To avoid multiple freeze-thaw cycles of your entire RNA sample, consider making smaller aliquots of it and storing those in -80°C.

Option #2 - TRIzol® Protocol

1. Homogenize or lyse tissues or cells in TRIzol® or a similar product.
   • For tissues: use 1 mL of TRIzol® per 100 mg of cells.
   • For cultured cells: use 1 mL of TRIzol® per 1 X 10⁷ cells.
2. Allow sample(s) to sit at room temperature for 5 minutes to allow for dissociation of the nucleoprotein complexes.
   • The effectiveness of your RNA isolation will depend on how effective your cell lysis protocol is. While simple homogenization is effective for most mammalian tissues; more hardy tissues such as bone, or bacteria/yeast/plant samples will require additional steps to effectively lyse open the cells.
3. Extract RNA from the homogenized sample(s). Add 0.2 mL of Chloroform/Isoamyl alcohol (49:1) per 1 mL of TRIzol® used. Shake vigorously by hand for 10 seconds.
4. Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at 12,000 × g at 4°C to separate RNA from the rest of the tissue/cell lysate.
   Pro-Tip
   Having your samples spin at 4°C helps reduce RNA degradation. If you don’t have access to a refrigerated centrifuge, you can carefully bring a centrifuge into a cold room for centrifugation. Once you’re done using the centrifuge, bring this equipment back to room temperature, as prolonged storage in the cold room may damage it.
5. Using a pipettor, transfer the top, aqueous phase to a new RNAse-free tube.
   • The mixture separates into a bottom organic layer, an interphase layer, and a top, aqueous layer. If using TRIzol®, the bottom layer will be a red-pink color. Take care to not disturb or collect the interphase layer with your pipette tip.
   Pro-Tip
   To collect as much of the top aqueous phase without disturbing the interphase layer, consider using a lower volume pipettor like a p200 to collect a majority of the aqueous phase. You may have to collect twice or more from the same tube, but unlike using a p1000 tip it will give you more control of where you’re aiming your tip in the tube.
6. Precipitate your sample(s). You can use either Isopropanol or lithium Chloride for this step.
   • Isopropanol (Option A): Add 1 volume of Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour.
   • Lithium Chloride (Option B): LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of 7.5 M LiCl solution to bring the concentration of LiCl in the extracted aqueous layer to 2.5 M. Incubate at -20°C for 1 hour.
   Pro-Tips
   If you anticipate your RNA yield to be small, RNase-free Glycogen may be used as a carrier to facilitate RNA precipitation. This does not affect the quality of RNA or downstream applications.
   
   To improve yield of RNA, instead of incubating at -20°C for 1 hour, you can try incubating at -80°C overnight.
7. Centrifuge for 20 minutes at 10,000 x g at 4°C and discard the supernatant. There should be a gel-like white pellet of total RNA in the bottom of the tube.
8. Wash the RNA by resuspending the pellet in 0.5–1 ml of 75% Ethanol and vortex for a few seconds. Centrifuge for 5 minutes at 10,000 x g at 4 °C and remove the supernatant.
9. Remove as much of the ethanol wash as possible without disturbing the pellet. Air-dry the pellet for 5-10 minutes.
   Critical
   It is important to not let the pellet get too dry before resuspending, as this affects the solubility of the RNA.
   Pro-Tip
   To prevent overdrying, watch the pellet and carefully remove any residual ethanol wash and add RNase-free water or TE as soon as the entire tube is dried but while the white pellet is still visible.
10. Resuspend RNA pellet in RNase-free water or TE. Quantify and assess the quality of your RNA sample(s) using a spectrophotometer (such as a Nanodrop), agarose gel, or bioanalyzer. For more information on nucleic acid quantification, see our protocol for DNA quantification, which can be modified for RNA.Store your RNA sample(s) at -80 °C to prevent RNA degradation and avoid multiple freeze-thaw cycles.
    Pro-Tip
    To avoid multiple freeze-thaw cycles of your entire RNA sample, consider making smaller aliquots of it and storing those in -80°C.