RNA Extraction

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The protocol for extracting RNA from tissue samples.

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• General tips for RNA extraction

• Homogenize tissue sample with TissueRuptor

Kits that we use

• TRI Reagent RNA Isolation

  • (Trizol based)

  • Works well with Crassostrea virginica

• Qiagen RNAeasy Kit

• ZYMO Quick DNA/RNA Miniprep

General tips for RNA extraction

• Work surfaces should be cleaned with 10% Bleach and then RNAase Away to ensure an RNAase free environment.

• RNA extraction is highly dependent on the starting tissue and quality of preservation. Only extract RNA from:

  • Fresh tissue

  • Tissue flash frozen in liquid Nitrogen

    • Some people recommend freezing tissues in RNAlater, so that when defrosting, RNA is still preserved

      • We have done this successfully, but have not formally tested it.

  • Tissue preserved in RNAlater

    • Note- it is best to put tissue in RNAlater, place in refrigerator overnight, and then freeze

• More general RNA tips can be found here and here

• Extractions need to be performed in the chemical hood

Homogenize tissue sample with TissueRuptor

The Tissue Ruptor uses disposable probes that can be sterilized and re-used.

To sterilize probes in the Autoclave:

• place probes in a large beaker and cover with aluminum and autoclave tape

• autoclave at 120°C for 30 min (20 min to reach temperature + 15 min at 120°C)

• Note TissueRuptor Probes may be cleaned /sterilized up to 5 times (15 min at 120°C) or until visible signs of wear are seen

Steps to homogenize:

• Get each tissue out of the freezer and keep on ice

  • do one tissue at a time

• prepare 2 mL vials (with somewhat flat bottoms) each with 600 µl DNA/RNA Lysis Buffer

• cut ~25 mg of tissue and put in prepared lysis vials

• keep vials on ice

• run TissueRuptor at level 3 for 7 seconds (or until tissue is completely lysed)

• return to ice

• clean probe with 3 washes of DI water

  • you should have 3 separate falcon tubes with DI water

  • run TissueRuptor through each DI wash for 5 seconds

  • remove probe and set aside to be autoclaved prior to re-use

  • secure a new sterilized probe to the TissueRuptor

Protocol for TRI Reagent RNA Isolation

Adapted from official protocol. Download here

Materials

• Nuclease-free Water

• Equipment for grinding/homogenizing solid tissue Appropriately sized RNase-free centrifuge tubes with secure closures, compatible with phenol/chloroform (polypropyl- ene, or polyallomer), and capable of withstanding centrifugal forces of 12,000 x g

• Centrifuge capable of 12,000 x g

• 1-bromo-3-chloropropane (BCP; recommended, e.g., MRC, Cat #BP 151)

• 100% isopropanol, ACS grade or better

• 100% ethanol, ACS grade or better

Procedure

1. Homogenize tissue samples in 10–20 volumes TRI Reagent solution.

   • Handling fresh tissue: Immediately after dissection, inactivate RNases by any one of the following treatments:

     • Homogenize in TRI Reagent solution immediately

     • Freeze rapidly in liquid nitrogen (tissue pieces must be small enough to freeze in a few seconds)

     • Submerge in a tissue storage buffer RNAlater®

   • Handling frozen tissue: Weigh frozen tissue, break into pieces smaller than ~50 mg (keeping tissue completely frozen), and homogenize directly in TRI Reagent solution. Larger pieces of tissue, very hard or fibrous tissues, and tissues with a high RNase content must typically be ground to a powder in liquid nitrogen for maximum RNA yield.

   • Homogenizing tissue: Homogenize samples in 10–20 volumes TRI Reagent solution (e.g., 1 mL TRI Reagent solution per 50–100mg tissue), using standard homogenization procedures.

     • The sample volume should not exceed 10% of the volume of TRI Reagent solution used for homogenization.

2. Incubate the homogenate for 5 min at room temp.

   • This 5 min room temperature incubation allows nucleoprotein complexes to completely dissociate.

   • Homogenized samples can be stored at –70°C for at least one month.

3. (Optional) Centrifuge at 12,000 xg for 10 min at 4°C and transfer the supernatant to a fresh tube.

   • This optional centrifugation is only required to remove insoluable material from homogenates that contain high amounts of protein, fat, polysaccharide, or extracellular material, such as muscle, fat tissue, and tuberous parts of plants. Centrifugation pellets, extracellular membranes, polysaccharides, and high molecular weight DNA, leaving the RNA in the supernatant. High molecular weight DNA can be recovered from the pellet by following the wash and solubilization steps of the DNA isolation protocol, available here

4. Add 100 μL BCP per 1 mL of TRI Reagent solution, mix well, and incubate at room temp for 5–15 min.

   • Add 100 μL BCP per 1 mL of TRI Reagent solution used for homogenization. Alternatively, 200 μL of chloroform (without isoamyl alcohol) can be used in place of BCP.

   • Cap the tubes tightly and shake vigorously for 15sec.

   • Incubate the mixture at room temperature for 5–15 min.

5. Centrifuge at 12,000 x g for 10–15 min at 4°C, then transfer the aqueous phase to a fresh tube.

   • Centrifuge at 12,000 x g for 10–15 min at 4°C.

   • Transfer the aqueous phase (colorless top layer) to a fresh tube.

   • RNA remains exclusively in the aqueous phase whereas DNA and protein are in the interphase and organic phase.

   • The interphase and lower, red, organic phase can be stored at 4°C for subsequent isolation of DNA and protein. A protocol is posted here

6. Add 500 μL of isopropanol per 1 mL of TRI Reagent solution, vortex for 5–10 sec, and incubate at room temp for 5–10 min.

   • Add 500 μL of isopropanol per 1 mL of TRI Reagent solution used for sample homogenization.

   • Vortex at moderate speed for 5–10 sec.

   • Incubate the samples at room temp for 5–10 min.

   • Note For oysters, we use a modified protocol:

     • Transfer the aqueous phase from step 5 to a fresh tube. For each 1 mL of TRI Reagent solution used for the homogenization, add 250 μL of isopropanol and 250 μL of a high salt precipitation solution (e.g. 0.8 M sodium citrate and 1.2 M NaCl).

     • Mix well, store for 5–10 min at room temperature, and centrifuge at 12,000 x g for 8 min at 4–25°C.

     • Wash the resulting RNA pellet as described in steps 8 and 9 of the procedure.

7. Centrifuge at 12,000 x g for 8 min at 4–25°C, and discard the supernatant.

   • Centrifuge at 12,000 x g for 8 min at 4–25°C.

   • Carefully remove the supernatant without disturbing the pellet.

     • Precipitated RNA forms a gel-like or white pellet on the side and bottom of the tube.

8. Add 1 mL of 75% ethanol per 1 mL of TRI Reagent solution.

   • Add 1 mL of 75% ethanol per 1 mL TRI Reagent solution used for sample homogenization to each sample to wash the RNA pellets.

9. Centrifuge at 7,500 x g for 5 min, remove the ethanol, and briefly air dry the RNA pellet.

   • Centrifuge at 7,500 x g for 5 min at 4–25°C.

     • If the precipitated RNA floats or does not form a compact pellet, repeat the centrifugation at 12,000 x g for 5 min to consolidate the pellet at the bottom of the tube.

   • Remove the ethanol wash without disturbing the pellet.

   • Remove all residual ethanol by centrifuging again briefly and removing the ethanol that collects with a fine tip pipette. Complete removal of ethanol is necessary for the RNA to perform well in downstream applications.

   • Air dry the RNA pellet for 3–5min.

     • Do not completely dry the RNA pellet as this will greatly decrease its solubility. Do not dry RNA by vacuum centrifugation. 10.Dissolve RNA in the buffer of your choice.

   • Dissolve RNA in THE RNA Storage Solution (P/N AM7000, AM7001), Nuclease-free Water, or your choice of buffer‡ by passing the solution a few times through a pipette tip or by vigorous vortexing

   • The resuspension volume is determined by the size of the RNA pellet. 3–5 mm pellets typically require 300–500 μL. If necessary, increase the resuspension volume or incubate at 55–60°C to completely dissolve the pellet.

   • Store at 4°C for immediate analysis. For long-term storage, store at –70°C or colder.

Protocol with Qiagen RNAeasy Kit

Reagents and supplies

• RNAeasy kit purification of total RNA from small amounts of starting material

• Molecular grade EtOH

Equipment

• Tissue homogenizer with replacable probes (Jon can you add specs of ours here)

• Micro centrifuge > 8,000 rpm

Notes before starting

• If purifying RNA from cell lines rich in RNases, or tissue, add either 10 μl β-mercaptoethanol (β-ME), or 20 μl 2 M dithiothreitol (DTT)*, to 1 ml Buffer RLT. Buffer RLT with β-ME or DTT can be stored at room temperature for up to 1 month.

• Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution.

• Remove RNAlater®-stabilized tissue from the reagent using forceps.

  Procedure

  1. Do not use more than 30 mg tissue. Disrupt the tissue and homogenize the lysate in the appropriate volume of Buffer RLT (350 μl for less than 20 mg; 650 μl for less than 30 mg). Centrifuge the lysate for 3 min at maximum speed. Carefully remove the supernatant by pipetting, and use it in step 2.

  2. Add 1 volume of 70% ethanol to the lysate, and mix well by pipetting. Do not centrifuge. Proceed immediately to step 3.

  3. Transfer up to 700 μl of the sample, including any precipitate, to an RNeasy Mini spin column placed in a 2 ml collection tube (supplied). Close the lid, and centrifuge for 15 s at ≥8000 x g. Discard the flow-through.

  4. Add 700 μl Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for 15 s at ≥8000 x g. Discard the flow-through.

  5. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 15 s at ≥8000 x g. Discard the flow-through.

  6. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 2 min at ≥8000 x g.

  7. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied). Centrifuge at full speed for 1 min to dry the membrane.

  8. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30–50 μl RNase-free water directly to the spin column membrane. Close the lid, and centrifuge for 1 min at ≥8000 x g to elute the RNA.

  9. If the expected RNA yield is >30 μg, repeat step 7 using another 30–50 μl of RNase-free water, or using the eluate from step 7 (if high RNA concentration is required). Reuse the collection tube from step 7.

Protocol for ZYMO Quick DNA RNA Miniprep and TissueRuptor homogenization

Protocol

Materials

• ZYMO Quick-DNA/RNA Miniprep

• TissueRuptor

• TissueRuptor Disposable Probes

• Ethanol (99%)

Equipment

• Centrifuge for 1.5 mL spin columns

Prep

• Sterilize TissueRuptor probes in the Autoclave:

  • place probes in a large beaker and cover with aluminum and autoclave tape

  • autoclave at 120°C for 35 min (20 min to reach temperature + 15 min at 120°C).

  • Note TissueRuptor Probes may be cleaned /sterilized up to 5 times (15 min at 120°C) or until visible signs of wear are seen

• Wipe of bench with 10% Bleach and RNAse Away

• Leave UV hood on for 1 hour to sterilize the working area

Procedure

1. Homogenize- TissueRuptor

   • Get each tissue out of the freezer and keep on ice

     • do one tissue at a time

   • prepare 2 mL vials (with somewhat flat bottoms) each with 600 µl DNA/RNA Lysis Buffer

   • cut ~25 mg of tissue and put in prepared lysis vials

   • keep vials on ice

   • run TissueRuptor at level 3 for 7 seconds (or until tissue is completely lysed)

   • return to ice

   • clean probe with 3 washes of DI water

     • you should have 3 separate falcon tubes with DI water

     • run TissueRuptor through each DI wash for 5 seconds

     • remove probe and set aside to be autoclaved prior to re-use

     • secure a new sterilized probe to the TissueRuptor

   • repeat steps with the next tissue

   • centrifuge samples at 14`,000 rfc x 30 seconds (may want to increase this)

2. Transfer the sample lysed in DNA/RNA Lysis Buffer into a SpinAway™ Filter (yellow) in a Collection Tube

   • centrifuge 16,000 rfc x 30 sec

   • Save the flow-through for RNA purification and the filter for DNA purification!

3. DNA/RNA Purification

   • 3a: DNA Purification (DNA is in the filter)

     • Transfer the Spin-Away Filter (yellow) into a new Collection Tube

   • 3b: RNA Purification(RNA is in the flow-through)

     • Add 600 μL volume ethanol (95-100%) to the flow-through (1:1 vol) and mix well.

     • Transfer the sample into a Zymo-Spin™ IICR Column in a Collection Tube

       • max 600 μL at a time

     • centrifuge 16,000 rfc x 30 sec

     • Discard the flow-through

4. Add 400 µl DNA/RNA Prep Buffer to the column

   • centrifuge 16,000 rfc x 30 sec

   • discard the flow-through

5. Add 700 µl DNA/RNA Wash Buffer to the column

   • centrifuge 16,000 rfc x 30 sec

   • Discard the flow-through.

6. Add 400 µl DNA/RNA Wash Buffer

   • centrifuge 16,000 rfc x 2 min to ensure complete removal of the wash buffer

   • Then carefully, transfer the column into a nuclease-free tube (not provided).

7. Elute DNA/RNA

   • 7a: DNA

     • add 50 µl DNase/RNase-Free Water

       • or 100 µl if you need it less concentrated

     • let stand 2-5 min

     • centrifuge 16,000 rfc x 30 sec

   • 7b: RNA

     • add 30 µl DNase/RNase-Free Water

       • or 50 µl if you need it less concentrated

     • centrifuge 16,000 rfc x 30 sec