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    Reliably Lyse Your Cells and Tissues to Get the RNA You Need

    Below are recipes to help you prepare your RNA extraction buffers and reagents. Typically, you’ll need to use mechanical or enzymatic shearing to speed up the process and avoid RNA degradation, but the reagents and materials you need depends on your specific project.

    If you’re just getting started on RNA extraction, read our Guide to RNA Extraction.

    You can find all of the RNA extraction reagents mentioned here at MP Bio. Additionally, you can explore our lysing matrices, RNA isolation kits, and ready-to-use RNA extraction buffers—available for virtually any sample type.

    Use Acid-Guanidium-Phenol Extraction for Cells and Tissues

    This method is useful for extracting total RNA or viral RNA, however it is generally unsuitable for purifying short RNAs such as siRNA, miRNA, gRNA, and tRNA.

    This is a liquid-liquid extraction technique that relies on phase separation of an aqueous sample mixed with a phenol-chloroform solution. Using guanidium thiocyanate, nucleic acids partition into the aqueous phase and proteins partition into the organic phenol phase. Using acidic conditions (pH 4-6), you can push DNA into the organic phase while retaining RNA in the aqueous phase (under neutral conditions, both DNA and RNA partition into the aqueous phase).

    The basic steps of this method include (reagents and recipes in the table below):

    1. Homogenize cells using cell lysis solution (and lysing matrix if needed)
    2. Transfer the homogenate to a fresh tube and add sodium acetate, water-saturated phenol, chloroform/isoamyl alcohol or bromochloropropane—mixing after each addition—and incubating at a cool temperature
    3. Centrifuge, and transfer the upper aqueous phase (this contains your RNA!) to a fresh tube
    4. Precipitate, wash, and resuspend the RNA

    RNA Extraction Reagents and Recipes

    Reagents and Recipes for Acid-Guanidium-Phenol RNA Extraction

    Step 1 Reagents and Recipes

    Cell Lysis Solution Final Concentration
    • 4 M guanidine thiocyanate
    • 25 mM sodium citrate
    • 0.5% Sarkosyl
    • 0.1 M 2-ME

    Stock solution
    • Mix
      • 293 ml water
      • 17.6 ml of 0.75 M sodium citrate, pH 7.0
      • 26.4 ml of 10% (w/v) N-lauroylsarcosine (Sarkosyl)
    • Add 250 g guanidine thiocyanate
    • Stir at 60° to 65°C to dissolve

    Store up to 3 months at room temperature

    Working solution (50 mL)
    • 50 ml of stock solution
    • Add 0.35 ml 2-mercaptoethanol (2-ME)
    • Store up to 1 month at room temperature

    Step 2 Reagents and Recipes

    2 M sodium acetate, pH 4
    • Add 16.42 g sodium acetate (anhydrous) to 40 ml water and 35 ml glacial acetic acid.
    • Adjust the solution to pH 4 with glacial acetic acid
    • Dilute to 100 ml with water.

    Store up to 1 year at room temperature

    Water-saturated phenol
    • Dissolve 100 g phenol crystals in water at 60° to 65°C
    • Remove the upper water phase

    Store up to 1 month at 4°C. Do not use buffered phenol in place of water-saturated phenol.

    49:1 (v/v) chloroform/isoamyl alcohol (or bromochloropropane)

    Step 3 Reagents and Recipes

    Centrifuge and transfer aqueous phase to a new tube—no reagents necessary

    Step 4 Reagents and Recipes

    Precipitation: 100% isopropanol

    Wash: 75% ethanol

    To 1 liter of 95% alcohol, add 294 ml DEPC water

    Resuspension: DEPC-treated water
    • Add 1 mL of fresh DEPC to 1 L of H2O
    • Shake well to disperse the DEPC through the H2O
    • Incubate at 37°C for at least 12 h and/or autoclave at 15 psi on liquid cycle for 20 min to inactivate the remaining DEPC

     

    RNA Extraction in Practice

    A variety of methods exist for extracting RNA and the equipment and RNA extraction reagents you need will depend on your sample type, the type of RNA you are aiming to isolate, and your downstream application.

    Read more about how Acid-Guanidium-Phenol Extraction works and how research groups are tailoring it to their specific needs: