SOP: Simultaneous DNA and RNA Extraction from Animal Tissues using QIAGEN AllPrep DNA/RNA Mini Kit

Applies to: Fresh or frozen animal tissues ≤30 mg per spin column Kit: QIAGEN AllPrep DNA/RNA Mini Kit (Cat. No. 80204) Location: __________

Document Control

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

Change History

Purpose

Simultaneous purification of genomic DNA and total RNA from the same tissue sample using the AllPrep DNA spin column (selective DNA binding in high-salt buffer) and the RNeasy spin column (silica membrane RNA binding). This enables paired DNA/RNA analysis from a single biopsy or tissue aliquot.

Scope

All personnel performing dual DNA/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.
• β-mercaptoethanol is toxic and has a strong odor. Handle in a fume hood.
• Use RNase-free tubes, tips, and reagents only. Change gloves frequently.
• Buffer RLT Plus contains guanidine isothiocyanate — harmful if swallowed or in contact with skin. Do not mix with bleach.

References

• QIAGEN AllPrep DNA/RNA Mini Handbook, November 2020.
• NASA GeneLab SOP GL-SOP-3.1 v1.0.

Materials and Equipment

Provided in kit (Cat. No. 80204, 50 preps)

• Buffer RLT Plus
• Buffer RW1
• Buffer RPE (concentrate — add ethanol before first use)
• Buffer AW1
• Buffer AW2
• AllPrep DNA Mini Spin Columns
• RNeasy Mini Spin Columns
• Collection Tubes (1.5 mL and 2 mL)
• RNase-free Water

Not supplied

• β-mercaptoethanol (β-ME)
• 96–100% ethanol
• 70% ethanol (freshly prepared)
• RNase-free DNase Set (Cat. No. 79254) — for on-column DNase treatment
• RNase/DNase-free 1.5 mL tubes
• Microcentrifuge capable of ≥12,000 × g
• Homogenization tools: rotor-stator homogenizer, or TissueLyser with stainless steel beads (3–7 mm), or RNase-free pestle, or mortar and pestle with liquid nitrogen
• Optional: RNase-free syringe with 20-gauge needle

Preparations (before each extraction session)

1. Prepare Buffer RPE (first use only)

   • Add 4 volumes of 96–100% ethanol to the concentrate. Mark the bottle to indicate ethanol has been added.

2. Prepare fresh Lysis Buffer

   • Add 10 µL β-mercaptoethanol per 1 mL Buffer RLT Plus. Mix well. Prepare only what you need; discard unused buffer.

3. Prepare DNase Mix (per sample)

   • Dissolve lyophilized DNase I (1500 Kunitz units) in 550 µL RNase-free water. Mix gently. Aliquot and store at −20 °C (stable up to 9 months; thawed aliquots stable at 2–8 °C for 6 weeks).
   • Per sample: 10 µL DNase I stock + 70 µL Buffer RDD. Mix by gentle inversion — do not vortex.

4. Pre-chill microcentrifuge to 4 °C if required for lysate clarification.

5. Heat 2 mL RNase/DNase-free water to 70 °C for DNA elution (optional, improves yield).

6. Input tissue mass

   Tissue type	Maximum input
   Standard soft tissue	≤30 mg
   Muscle, heart, skin	≤15 mg (LN₂-preserved) or ≤10 mg (RNAlater)
   Liver	≤20 mg

Procedure

1. Tissue Disruption and Homogenization

1. Weigh tissue (≤30 mg). Place in a pre-chilled RNase-free tube on ice.

2. Add 600 µL Buffer RLT Plus (with β-ME).

3. Homogenize immediately using one of the following:

   Option A — Rotor-stator: Homogenize for 30–45 s until no visible tissue pieces remain.

   Option B — TissueLyser / bead mill: Add a single 5 mm stainless steel bead. Run at 25 Hz for 2 × 2 min (rotate adapter halfway).

   Option C — Mortar & pestle with liquid nitrogen: Grind tissue to a fine powder under LN₂. Transfer powder to a tube, let LN₂ evaporate, then add Buffer RLT Plus and vortex.

   Option D — Needle and syringe: Pass lysate through a 20-gauge needle at least 5–10 times until homogeneous.

4. Centrifuge the lysate at full speed (≥12,000 × g) for 3 min, RT.

5. Carefully transfer the supernatant to a new tube. Do not disturb the pellet.

2. Bind Genomic DNA

1. Transfer the cleared lysate onto an AllPrep DNA Mini Spin Column in a 2 mL collection tube.
2. Centrifuge at ≥8,000 × g for 30 s, RT.
3. Set aside the DNA column at 4 °C (or RT for up to 1 h) — proceed to RNA purification first.
4. Save the flow-through for RNA isolation.

3. Adjust RNA Binding Conditions

1. Add 1 volume of freshly prepared 70% ethanol to the flow-through (e.g. 600 µL ethanol to 600 µL flow-through). For liver tissue, use 50% ethanol instead.
2. Mix well by pipetting. Do not centrifuge. A precipitate may form — this is normal; proceed immediately.

4. Bind and Wash RNA

1. Transfer up to 700 µL of the sample onto an RNeasy Mini Spin Column in a 2 mL collection tube.
2. Centrifuge at ≥8,000 × g for 15 s, RT. Discard flow-through.
3. Repeat until all sample has passed through the column.
4. Add 350 µL Buffer RW1. Centrifuge at ≥8,000 × g for 15 s, RT. Discard flow-through.

5. On-Column DNase Treatment

1. Pipette 80 µL DNase Mix (10 µL DNase I + 70 µL Buffer RDD) directly onto the membrane.
2. Incubate at RT for 15–30 min (30 min recommended for maximum removal).
3. Add 350 µL Buffer RW1. Centrifuge at ≥8,000 × g for 15 s, RT. Discard flow-through.

6. Wash and Dry RNA Column

1. Replace the collection tube with a new 2 mL tube.
2. Add 500 µL Buffer RPE (ethanol added). Centrifuge at ≥8,000 × g for 15 s, RT. Discard flow-through.
3. Add another 500 µL Buffer RPE. Centrifuge at ≥8,000 × g for 2 min, RT, to dry the membrane.
4. Optional: place column in a new 2 mL tube and centrifuge at full speed for 1 min to eliminate residual ethanol.

7. Elute RNA

1. Place the RNeasy column in a new 1.5 mL collection tube.
2. Add 30–50 µL RNase-free water directly onto the membrane.
3. Incubate 1–5 min at RT.
4. Centrifuge at ≥8,000 × g for 1 min, RT.
5. Optional: repeat with a second 30 µL RNase-free water for maximum yield. Pool eluates if the second elution is ≥30% of the first.
6. Place RNA on ice immediately.

8. Wash Genomic DNA Column

1. Retrieve the AllPrep DNA column from step 2.
2. Add 500 µL Buffer AW1. Centrifuge at ≥8,000 × g for 15 s, RT. Discard flow-through.
3. Add 500 µL Buffer AW2. Centrifuge at full speed (≥14,000 × g) for 2 min, RT, to dry the membrane.
4. Optional: place column in a new 2 mL tube and centrifuge at full speed for 1 min to eliminate residual ethanol.

9. Elute Genomic DNA

1. Place the AllPrep DNA column in a new 1.5 mL collection tube.
2. Add 50–100 µL RNase/DNase-free water (pre-warmed to 70 °C for improved yield) directly onto the membrane.
3. Incubate 2 min at RT.
4. Centrifuge at ≥8,000 × g for 1 min, RT.
5. Optional: repeat with a second 30 µL for maximum yield.

10. Quantification and Quality Assessment

RNA:

• Measure concentration by Qubit RNA assay or NanoDrop A260.
• Record A260/280 (expect ≥1.8) and A260/230 (expect ≥1.5) ratios.
• Assess integrity by Fragment Analyzer, Bioanalyzer, or TapeStation (target RQN/RIN ≥7 for most applications).

DNA:

• Measure concentration by Qubit dsDNA assay or NanoDrop A260.
• Record A260/280 (expect ~1.8) and A260/230 ratios.
• Assess fragment size by TapeStation Genomic DNA assay or agarose gel if required.

11. Storage

• RNA: Keep on ice for immediate use. Store at −80 °C for long term.
• DNA: Store at −20 °C (short term) or −80 °C (long term). Avoid repeated freeze-thaw cycles.

Special Considerations for Muscle, Heart, and Skin Tissues

These tissues are dense and fibrous, leading to potential column clogging and lower yields.

• Start with ≤15 mg (LN₂-preserved) or ≤10 mg (RNAlater-preserved).
• Pre-warm Buffers RW1 and RPE to 37 °C for 30 min before use.
• Centrifuge lysate twice at full speed to minimize cell debris.
• Load a maximum of 400 µL per column pass.
• If the column clogs: incubate wash solution on column for 5 min at 37 °C before centrifuging.
• Elute with pre-warmed (37 °C) water in two passes (50 µL + 30 µL); combine if second elution is ≥30% of first.
• If yields remain low: process two 15 mg aliquots separately and combine eluates.

Acceptance and Rejection Criteria

• Elution volume and expected yield range documented per tissue type.
• No visible carryover of debris in eluate.
• Proceed if spectrophotometric and integrity checks meet project-specific thresholds.

Expected Yields

Troubleshooting

Waste Disposal

• Collect β-mercaptoethanol and guanidine-containing wastes according to institutional chemical waste procedures.
• Dispose of biological materials under local biosafety rules.
• Spin columns with bound nucleic acids are non-hazardous after elution.

Records to Maintain

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

End of SOP