SOP: Total RNA Extraction from Animal Tissues using PureLink™ RNA Mini Kit

Applies to: Fresh or frozen animal tissues ≤200 mg per spin cartridge Kit: PureLink™ RNA Mini Kit (Cat. No. 12183020) Location: __________

Document Control

• SOP ID: __________
• Version: 1.0
• Effective Date: __________
• Review Due: __________
• Author: __________
• Approved By: __________

Change History

Purpose

Standardized procedure to isolate high-quality total RNA from animal tissues using silica spin columns.

Scope

All personnel performing RNA extraction from animal tissues in this laboratory.

Responsibilities

• Operators: Follow this SOP, record batch details, and maintain RNase-free practice.
• Supervisors: Ensure training, PPE compliance, and kit maintenance.

Safety and RNase-Free Practice

• Wear lab coat, gloves, and eye protection.
• Work in a clean area. Treat benches and tools with RNase decontamination solution.
• 2‑mercaptoethanol is toxic and has a strong odor. Handle in a fume hood.
• Use RNase‑free tubes, tips, and reagents only. Change gloves frequently.

References

• PureLink™ RNA Mini Kit Quick Reference and User Guide.

Materials and Equipment

Provided in kit

• Lysis Buffer
• Wash Buffer I
• Wash Buffer II
• Spin Cartridges with Collection Tubes
• Additional Collection Tubes
• Recovery Tubes
• RNase‑free Water

Not supplied

• 2‑mercaptoethanol
• 96–100% ethanol
• PBS
• (Optional) PureLink™ DNase Set (Cat. No. 12185010)
• RNase‑free 1.5 mL and 15 mL tubes
• Microcentrifuge capable of 12,000 × g
• Homogenization tools: rotor‑stator homogenizer; RNase‑free pestle; mortar and pestle with liquid nitrogen; or 1 mL RNase‑free syringe with 18–21 G needle

Preparations

1. Prepare Wash Buffer II

   • Add 16 mL 96–100% ethanol to the bottle labeled Wash Buffer II. Mark the bottle to indicate ethanol added. Store at room temperature.

2. (Optional) Prepare DNase Mix Prepare 80 µL per sample in an RNase‑free tube and store at −20 °C:

   • 10X DNase I Reaction Buffer: 8 µL
   • DNase I (~3 U/µL): 10 µL
   • RNase‑free water: 62 µL

3. Prepare fresh Lysis Buffer (each use)

   • Add 10 µL 2‑mercaptoethanol per 1 mL Lysis Buffer required. Mix. Prepare only what you need.

4. Plan input and Lysis Buffer volume

   Tissue amount	Lysis Buffer per sample
   ≤10 mg	0.3 mL (use 0.6 mL if using a rotor‑stator)
   10–30 mg	0.6 mL
   30–200 mg	0.6 mL per 30 mg

5. Capacity note

   • If a tissue contains >1 mg total RNA, split across multiple spin cartridges.

Recommended Homogenization Setups

Procedure

1. Tissue Disruption and Homogenization

Keep samples on ice after harvesting. Add pre‑mixed Lysis Buffer with 2‑ME per table above.

Option A. Microfuge pestle

1. Place tissue in a pre‑chilled RNase‑free tube on ice. Add Lysis Buffer.

2. Mince with RNase‑free pestle until fully lysed.

3. For ≤100 mg fresh soft tissue: centrifuge ~1,200 × g, 2 min, RT. Transfer supernatant to a clean RNase‑free tube.

4. Further homogenize at RT using one of:

   • Pass lysate through a column homogenizer inserted in a collection tube; spin 12,000 × g, 2 min. Remove homogenizer.
   • Or pass lysate 5–10× through an 18–21 G needle and syringe; spin 12,000 × g, 2 min; transfer supernatant.

Option B. Rotor‑stator

1. Place tissue in pre‑chilled tube on ice. Add Lysis Buffer.

2. Homogenize:

   • ≤100 mg fresh soft tissue: ≥45 s.
   • Other tissue types: 30–40 s.

3. Clarify: centrifuge ~2,600 × g, 5 min, RT. Transfer supernatant.

Option C. Mortar & pestle with liquid nitrogen

1. Grind tissue to a fine powder in LN₂.
2. Transfer powder to a 2 mL RNase‑free tube and let LN₂ evaporate.
3. Add Lysis Buffer. Homogenize at RT using either the column homogenizer + 12,000 × g, 2 min spin or the syringe/needle method described above; then transfer supernatant.

2. Bind RNA

1. To the clarified lysate, add 1.5 volumes of 100% ethanol. Vortex to mix and dissolve any precipitate.
2. Load up to 700 µL onto the spin cartridge seated in a collection tube.
3. Centrifuge 12,000 × g, 15 s, RT. Discard flow‑through. Reinsert cartridge into the same collection tube.
4. Repeat loading/spin until the entire sample is processed.

3. Wash RNA

Choose A. No DNase or B. On‑column DNase.

A. Without DNase treatment

1. Add 700 µL Wash Buffer I. Spin 12,000 × g, 15 s.
2. Discard flow‑through and replace the collection tube.
3. Add 500 µL Wash Buffer II (ethanol added). Spin 12,000 × g, 15 s.
4. Discard flow‑through. Reinsert cartridge in the same tube.
5. Repeat Step 3 once more (second 500 µL Wash Buffer II, 15 s).

B. With on‑column DNase treatment

1. Add 350 µL Wash Buffer I. Spin 12,000 × g, 15 s.
2. Discard flow‑through and replace the collection tube.
3. Apply 80 µL DNase Mix directly to the membrane surface. Incubate 15 min, RT.
4. Add 350 µL Wash Buffer I. Spin ~2,600 × g, 5 min.
5. Discard flow‑through and replace the collection tube.
6. Add 500 µL Wash Buffer II (ethanol added). Spin 12,000 × g, 15 s.
7. Discard flow‑through. Reinsert cartridge in the same tube.
8. Repeat Step 6 once more (second 500 µL Wash Buffer II, 15 s).

4. Elute RNA

1. Dry‑spin the cartridge with collection tube 12,000 × g, 1 min. Discard the collection tube.
2. Place the cartridge into a recovery tube.
3. Add 30–100 µL RNase‑free water to the center of the membrane. Optional serial elution: up to 3 × 100 µL; pool eluates.
4. Incubate 1 min, RT.
5. Centrifuge 12,000 × g, 2 min, RT.

5. RNA Quantification and Quality

• Measure concentration by A260 or fluorometric RNA assay.
• Record A260/280 and A260/230 ratios.
• Assess integrity by electrophoresis or Bioanalyzer/TapeStation, if required.

6. Storage

• Keep on ice for immediate use.
• Store at −80 °C for long term.

Acceptance and Rejection Criteria

• Elution volume and expected yield range documented for each tissue type.
• No visible carryover of debris.
• Proceed if spectrophotometric and integrity checks meet project‑specific thresholds.

Troubleshooting Notes

• Low yield: increase elution volume or perform a second elution; confirm ethanol added to Wash Buffer II; ensure complete lysis and thorough homogenization.
• Column clogging: reduce input mass, clarify lysate, use needle‑syringe or column homogenizer.
• Genomic DNA contamination: include on‑column DNase step.

Waste Disposal

• Collect 2‑mercaptoethanol and ethanol wastes according to institutional chemical waste procedures.
• Dispose of biological materials under local biosafety rules.

Records to Maintain

• Date, operator, sample ID, tissue type and mass, homogenization method, reagent lot numbers, elution volume(s), concentration, purity ratios, integrity assessment, and storage location.
• Deviations and corrective actions.

Appendices

A. Quick Selection of Homogenization Method

• See table in Recommended Homogenization Setups above.

B. Input Mass and Lysis Buffer Planning

• See Preparations, Step 4 table.

End of SOP