TITLE: RNA Extraction Without a Kit
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes RNA extraction without a kit. To see the full abstract and additional resources, visit </div><div class = "text-block"><a href="https://www.addgene.org/protocols/kit-free-rna-extraction" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/kit-free-rna-extraction/</span></a></div></div>

[STEPS]
?. [Homogenization/Lysis]
Homogenize or lyse tissues or cells in Solution D.
For tissues: use   of Solution D per   of cells. For cultured cells: use   of Solution D per 1 X 107 cells.
?. [Homogenization/Lysis]
Allow sample(s) to sit at  for  to allow for dissociation of the nucleoprotein complexes.
0 Room temperature
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.
?. [Extraction]
Extract RNA from the homogenized sample(s). Transfer tissue/cell lysate to a  tube. Add the following sequentially to  of lysate: Add  of  sodium acetate, mix thoroughly by inversion. Add  water-saturated phenol, mix thoroughly by inversion. Add  of chloroform/isoamyl alcohol (49:1) and then shake vigorously by hand for .
4 ml
1 ml
0.1 ml
1 ml
0.2 ml
?. [Extraction]
Incubate sample(s) for  and centrifuge the sample(s) at to separate RNA from the rest of the tissue/cell lysate.
on ice
Centrifuge: 12000 34, 15 min, 4 10
*Pro-Tip*Having your samples spin at   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  , as prolonged storage in the cold room may damage it.
?. [Extraction]
Using a pipettor, carefully 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.
*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.
?. [Precipitation and Resuspension]
Wash the RNA by resuspending the pellet in – of 75% ethanol and vortex for a few seconds. Centrifuge at  and remove the supernatant.
0.5 ml
1 ml
Centrifuge: 10000 34, 5 min, 4 10
?. [Precipitation and Resuspension]
Remove as much of the ethanol wash as possible without disturbing the pellet. Air-dry the pellet for -.
*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.
?. [Precipitation and Resuspension]
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 on DNA quantification, which can be modified for RNA. Store your RNA sample(s) at  to prevent RNA degradation and avoid multiple freeze-thaw cycles.
-80 °C
*Pro-Tip* To avoid multiple freeze-thaw cycles of your entire RNA sample, consider making smaller aliquots of your original sample and storing those in  .
?. [Homogenization/Lysis]
If you are using Solution D, start with step-case 'Solution D'. If you are using TRIzol®, TRI Reagent®, RNAzol®, or QIAzol®, start with step-case 'TRIzol®'See the Materials section for recipes and reagents.
?. [Homogenization/Lysis]
Homogenize cells in TRIzol® or a similar product.
For tissues: use   of TRIzol® per   of cells.For cultured cells: use   of TRIzol® per 1 X 107 cells.
?. [Homogenization/Lysis]
Allow sample(s) to sit at  for  to allow for dissociation of the nucleoprotein complexes.
0 Room temperature
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.
?. [Extraction]
Extract RNA from the homogenized sample(s). Add  of chloroform/isoamyl alcohol (49:1) per  of TRIzol® used. Shake vigorously by hand for .
0.2 ml
1 ml
?. [Extraction]
Incubate sample(s) for - and centrifuge the sample(s) at to separate RNA from the rest of the tissue/cell lysate.
on ice
Centrifuge: 12000 34, 15 min, 4 10
*Pro-Tip*Having your samples spin at   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  , as prolonged storage in the cold room may damage it.
?. [Extraction]
Using a pipettor, carefully 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.
*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.
?. [Precipitation and Resuspension]
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   for  .
Lithium Chloride (Option B) - LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of   LiCl solution to bring the concentration of LiCl in the extracted aqueous layer to  . Incubate at   for  .
*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. To improve yield of RNA, instead of incubating at   for  , you can try incubating at    .
?. [Precipitation and Resuspension]
Centrifuge at  and discard the supernatant. There should be a gel-like white pellet of total RNA in the bottom of the tube.
Centrifuge: 10000 34, 20 min, 4 10
?. [Precipitation and Resuspension]
Wash the RNA by resuspending the pellet in – of 75% ethanol and vortex for a few seconds. Centrifuge at  and remove the supernatant.
0.5 ml
1 ml
Centrifuge: 10000 34, 5 min, 4 10
?. [Precipitation and Resuspension]
Remove as much of the ethanol wash as possible without disturbing the pellet. Air-dry the pellet for -.
*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.
?. [Precipitation and Resuspension]
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 on DNA quantification, which can be modified for RNA. Store your RNA sample(s) at  to prevent RNA degradation and avoid multiple freeze-thaw cycles.
-80 °C
*Pro-Tip* To avoid multiple freeze-thaw cycles of your entire RNA sample, consider making smaller aliquots of your original sample and storing those in  .
?. [Precipitation and Resuspension]
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   for  .
Lithium Chloride (Option B) - LiCl selectively precipitates RNA versus DNA or proteins. Add the correct amount of   LiCl solution to bring the concentration of LiCl in the extracted aqueous layer to  . Incubate at   for  .
*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. To improve yield of RNA, instead of incubating at   for  , you can try incubating at    .
?. [Precipitation and Resuspension]
Centrifuge at  and discard the supernatant. There should be a gel-like white pellet of total RNA in the bottom of the tube.
Centrifuge: 10000 34, 20 min, 4 10