RNA Extraction Without a Kit

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.

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.
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.
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.
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.

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.

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-Tip* 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.

*Pro-Tip* To improve yield of RNA, instead of incubating at -20°C for 1 hour, you can try incubating at -80°C overnight.

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.
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.
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.

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

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.
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.
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.
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.

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.

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-Tip* 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.

*Pro-Tip* To improve yield of RNA, instead of incubating at -20°C for 1 hour, you can try incubating at -80°C overnight.

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.
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.
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.

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.