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Soil RNA Extraction — RNeasy PowerSoil Total RNA

Bench manual for soil total RNA extraction using the QIAGEN RNeasy PowerSoil Total RNA Kit.

Input≤2 g soil
Final volume100 µL
HazardPhenol/chloroform
Elapsed time~2.5–3.5 h

Before Starting

Chemical hazard — phenol/chloroform/isoamyl alcohol

This protocol requires 3.5 mL phenol/chloroform/isoamyl alcohol (pH 6.5–8.0), user-supplied. Work in a chemical fume hood. Wear appropriate PPE (gloves, lab coat, eye protection). Dispose of phenol/chloroform waste according to your institution's hazardous waste policy.

Notes

  • Perform all centrifugation steps at room temperature (15–25°C).
  • Wear RNase-free gloves at all times and decontaminate the work area with RNase removal solution before starting.
  • Store the kit at room temperature (15–25°C) until the expiry date.

Materials

Kit Solutions

  • PowerBead Solution
  • Solution SR1
  • Solution IRS
  • Solution SR3
  • Solution SR4
  • Solution SR5
  • Solution SR6 (elution)
  • Solution SR7 (resuspension)

User-Supplied

  • Phenol/chloroform/isoamyl alcohol, pH 6.5–8.0, 3.5 mL per sample

Kit Hardware

  • 15 mL PowerBead Tube
  • 15 mL Collection Tubes
  • 2.2 mL Collection Tubes
  • JetStar Mini Column

Equipment

  • Vortex mixer with Vortex Adapter (cat. no. 13000-V1-15)
  • Centrifuge capable of 2500 × g and 13,000 × g
  • Chemical fume hood
  • Heat block or water bath at 45°C (if pellet is difficult to resuspend)

Part A: Bead Beating & Phase Separation

~45–60 min

  • Add up to 2 g soil to the 15 mL PowerBead Tube.
  • Add 2.5 mL PowerBead Solution, 0.25 mL Solution SR1, and 0.8 mL Solution IRS.
  • Add 3.5 mL phenol/chloroform/isoamyl alcohol (pH 6.5–8.0) in the fume hood. Cap and vortex until the biphasic layer disappears.
  • Place the PowerBead Tube on the Vortex Adapter and vortex at maximum speed for 15 min.
  • Centrifuge at 2500 × g for 10 min.
  • Transfer the upper aqueous phase to a clean 15 mL Collection Tube. Avoid the interphase and lower phenol layer. Discard the phenol/chloroform/isoamyl alcohol as hazardous waste. Note: In high-organic-matter soils, the biphasic layer may be firm and need to be pierced to remove the bottom phenol layer.

Part B: Precipitation & Cleanup

~55–70 min

  • Add 1.5 mL Solution SR3 to the aqueous phase. Vortex to mix. Incubate at 2–8°C for 10 min. Centrifuge at 2500 × g for 10 min at room temperature.
  • Transfer the supernatant (without disturbing the pellet) to a new 15 mL Collection Tube.
  • Add 5 mL Solution SR4. Invert or vortex to mix. Incubate at room temperature for 30 min.
  • Centrifuge at 2500 × g for 30 min.
  • Decant the supernatant. Invert the Collection Tube on a paper towel for 5 min.
  • Shake Solution SR5 to mix. Add 1 mL Solution SR5 to the tube. Resuspend the pellet completely by pipetting or vortexing. If difficult to resuspend, heat at 45°C for 10 min, then vortex. Repeat until fully dissolved.

Part C: Column Purification

~30–40 min

  • Prepare one JetStar Mini Column per sample: remove cap from a 15 mL Collection Tube and place the JetStar Mini Column inside so it hangs in the tube.
  • Add 2 mL Solution SR5 to the JetStar Mini Column. Allow to gravity flow completely. Do not let the column dry out before loading sample.
  • Load the RNA isolation sample (from Part B Step 6) onto the JetStar Mini Column. Allow to gravity flow through.
  • Add 1 mL Solution SR5 to the column. Allow to gravity flow through completely.
  • Transfer the JetStar Mini Column to a new 15 mL Collection Tube. Shake Solution SR6 to mix. Add 1 mL Solution SR6 to the column to elute bound RNA. Allow to gravity flow.

Part D: RNA Precipitation & Resuspension

~35–50 min

  • Transfer the eluted RNA to a 2.2 mL Collection Tube. Add 1 mL Solution SR4. Invert at least once to mix. Incubate at −15°C to −30°C for ≥10 min.
  • Centrifuge at 13,000 × g for 15 min to pellet RNA.
  • Decant the supernatant. Invert the 2.2 mL Collection Tube onto a paper towel for 10 min to air dry the pellet.
  • Resuspend the RNA pellet in 100 µL Solution SR7. RNA is ready for downstream use or storage.

Storage

Store RNA at −80°C for long-term use. Use −20°C only as a short-term fallback. Avoid repeated freeze-thaw cycles. Quantify with Qubit RNA HS/BR or equivalent before use.

Bench Record

Date
Operator
Sample IDs
Soil input per sample (g)
Phenol/chloroform lot #
SR4 incubation temperature−15 to −30°C
Final resuspension volume100 µL
Quantification method
RNA concentration (ng/µL)
A260/A280
Notes

Stop Points

Stop and ask before:

  • Continuing if you cannot see a clear phase separation after bead beating (Step A-6).
  • Discarding the pellet in Part B if you are unsure which layer is the pellet.
  • Proceeding to downstream cDNA synthesis if RNA concentration is much lower than expected or A260/A280 < 1.8.