RNeasy FFPE Handbook

Introduction

The RNeasy FFPE Kit is designed for the purification of total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. It enables the recovery of usable RNA fragments longer than 70 nucleotides, suitable for applications such as RT-PCR. FFPE samples often contain fragmented and chemically modified nucleic acids due to fixation and embedding processes. This kit is optimized to reverse formaldehyde modifications while minimizing RNA degradation. It is recommended for applications requiring fragmented RNA, and for cDNA synthesis, random or gene-specific primers should be used instead of oligo-dT primers. Staining protocols, such as cresyl violet, may impact RNA quality but have shown minimal impact on downstream RT-PCR analysis.

Principle and Procedure

The RNeasy FFPE procedure utilizes RNeasy technology for RNA purification. It involves:

• Removal of paraffin from FFPE tissue sections.
• Incubation in an optimized lysis buffer with proteinase K to release RNA.
• A short incubation at higher temperature to partially reverse formalin crosslinking, improving yield and quality.
• DNase treatment to remove DNA contamination.
• Binding of total RNA to an RNeasy MinElute spin column membrane.
• Washing away contaminants.
• Elution of purified RNA in RNase-free water.

Solutions for FFPE Research & Automation

QIAGEN offers dedicated products for FFPE samples, facilitating deparaffinization and efficient recovery of DNA, RNA, miRNA, and protein. Automation of FFPE sample preparation is available using the QIAcube Connect system. Key benefits of QIAGEN's FFPE portfolio include:

• Maximum data output with minimal sample consumption.
• Technologies that reverse crosslinks for higher yields.
• DNA, RNA, and protein purification without compromising analyte integrity.
• Optimized chemistries for analyzing lower quality FFPE analytes.
• Reliable FFPE research data.

RNA purification can be fully automated on QIAcube Connect or the classic QIAcube instruments, which process QIAGEN spin columns and integrate automated sample preparation into laboratory workflows. These instruments are preinstalled with various purification protocols, and additional protocols are available for download.

RNeasy FFPE Procedure Overview

The workflow involves the following steps:

• FFPE tissue sections are processed.
• Paraffin removal and drying is performed.
• Samples are lysed with proteinase K digestion followed by heat treatment.
• The supernatant is treated with DNase, then Buffer RBC and ethanol are added.
• Total RNA binds to the RNeasy MinElute column.
• The column is washed.
• Purified RNA is eluted.

Equipment and Reagents to Be Supplied by User

Users must supply the following:

• Sterile, RNase-free pipet tips
• 1.5 ml or 2 ml centrifuge tubes
• Microcentrifuge (with rotor for 2 ml tubes)
• Vortexer
• 100% ethanol
• Deparaffinization Solution (cat. no. 19093) or alternative reagent (e.g., heptane, xylene, limonene, CitriSolv)
• Disposable gloves
• Heating block or water bath capable of incubation at 80°C

Note: Always wear a suitable lab coat, disposable gloves, and protective goggles when working with chemicals.

Important Notes

Starting Material Considerations:

• Use tissue samples < 5 mm thick for formalin penetration.
• Fixate samples in 4-10% neutral-buffered formalin as soon as possible after surgical removal.
• Limit fixation time to a maximum of 24 hours to avoid over-fixation and severe nucleic acid fragmentation.
• Thoroughly dehydrate samples before embedding.
• Use low-melting paraffin for embedding.
• Starting material should be freshly cut FFPE tissue sections, up to 20 µm thick. Thicker sections may yield less RNA. Up to 4 sections (up to 10 µm thick, 250 mm² surface area) can be combined. More can be combined if total thickness is ≤ 40 µm or if tissue area is < 30% of the surface.
• For tissues with high DNA content (e.g., thymus), use fewer sections per preparation to avoid DNA contamination.
• If starting material is unknown, begin with no more than 2 sections per preparation.
• For materials other than microtome sections (e.g., core punches), refer to www.qiagen.com/goto/RNeasyFFPE.
• Do not overload the RNeasy MinElute spin column to maintain RNA yield and quality.

Preparation of Buffers

DNase I Stock Solution:

Dissolve lyophilized DNase I (1500 Kunitz units) in 550 µl RNase-free water using a needle and syringe. Mix gently by inverting the vial; do not vortex. Store aliquots at -30°C to -15°C for up to 9 months.

Buffer RPE:

Add 4 volumes (44 ml) of ethanol (96-100%) to the 11 ml Buffer RPE concentrate. Mix by shaking before use.

Protocol: Purification of Total RNA from FFPE Tissue Sections

Important points before starting:

• Read "Important Notes" (page 10) and Appendix B (page 25) if new to RNA work.
• Buffer RBC contains guanidine salt and is incompatible with bleach.
• Perform steps at room temperature (15–25°C) and work quickly.
• Use a microcentrifuge set at 15–25°C.
• Volumes indicated by ▲ are for 1-2 sections/sample; volumes indicated by ● are for >2 sections/sample.

Things to do before starting:

• Reconstitute Buffer RPE and RNase-Free DNase I if necessary.
• Equilibrate buffers to room temperature (15–25°C).
• Set heating blocks/water baths to 56°C and 80°C.

Procedure Summary:

1. Trim excess paraffin from the sample block.
2. Cut sections (5–20 µm thick). Discard first 2-3 sections if exposed to air.
3. Place sections in a 1.5 ml or 2 ml microcentrifuge tube.
4. Add 160 µl or 320 µl Deparaffinization Solution, vortex, centrifuge briefly.
5. Incubate at 56°C for 3 min, then cool to room temperature.
6. Add 150 µl or 240 µl Buffer PKD, mix by vortexing.
7. Centrifuge for 1 min at 11,000 x g.
8. Add 10 µl proteinase K to the lower phase, mix gently.
9. Incubate at 56°C for 15 min, then at 80°C for 15 min. (Crucial for crosslink reversal).
10. Transfer the supernatant to a new tube.
11. Incubate on ice for 3 min, then centrifuge for 15 min at 20,000 x g.
12. Transfer supernatant to a new tube.
13. Add DNase Booster Buffer and 10 µl DNase I stock solution, mix gently.
14. Incubate at room temperature for 15 min.
15. Add 320 µl or 500 µl Buffer RBC, mix.
16. Add 720 µl or 1200 µl ethanol (100%), mix.
17. Transfer 700 µl sample to an RNeasy MinElute spin column, centrifuge at ≥8000 x g for 15 s. Discard flow-through. Repeat for entire sample.
18. Add 500 µl Buffer RPE to the column, centrifuge at ≥8000 x g for 15 s. Discard flow-through.
19. Add 500 µl Buffer RPE again, centrifuge at ≥8000 x g for 2 min to wash the membrane. Discard collection tube.
20. Place column in a new 2 ml collection tube, centrifuge at full speed for 5 min to dry the membrane.
21. Place column in a new 1.5 ml collection tube. Add 14–30 µl RNase-free water directly to the membrane, centrifuge for 1 min at full speed to elute RNA.

Protocol: Purification of Total RNA from Microdissected FFPE Tissue Sections

This protocol is for total RNA purification from microdissected FFPE samples, which present challenges due to small starting material amounts and potential fixation/staining effects on RNA integrity. Cresyl violet staining has minimal impact on RNA quality. This protocol is not for microdissected cryosections; the RNeasy Plus Micro Kit is recommended for those. Equipment for sectioning, staining, and microdissection is available from Leica and P.A.L.M. Microlaser Technologies.

The procedure follows a similar logic to the standard FFPE protocol but is adapted for smaller sample volumes. Key steps include:

• After microdissection, place sample in a collection vessel with Buffer PKD (volume depends on vessel, e.g., 65 µl for Leica, 150 µl for others).
• If necessary, transfer to a larger tube and adjust volume to 150 µl with Buffer PKD.
• Add 10 µl proteinase K, mix, and incubate at 56°C for 15 min, then 80°C for 15 min.
• Incubate on ice for 3 min, then centrifuge at 20,000 x g for 15 min.
• Proceed with steps similar to the standard FFPE protocol (e.g., adding DNase, Buffer RBC, ethanol, binding to column, washing, and elution), often using the lower volume options (indicated by ▲).

Troubleshooting Guide

This guide addresses common issues:

• Clogged RNeasy MinElute spin column: Reduce starting material or ensure proper centrifugation temperature (15–25°C), warming sample to 37°C if needed.
• Low RNA yield: Use high-quality starting material (freshly fixed, not stored for too long, not mounted on slides for extended periods). Avoid overloading the column. Ensure sufficient deparaffinization. Repeat elution with a benchtop incubation.
• Low A260/A280 value: Use 10 mM Tris-Cl, pH 7.5, not water, for dilution before measurement.
• DNA contamination: For tissues with high DNA content, use fewer sections. Consider kits with integrated DNA removal for RT-PCR.
• RNA performance issues: Ensure the 80°C incubation is maintained for the full 15 minutes for crosslink reversal. Use random or gene-specific primers for cDNA synthesis. Keep amplicons short. Ensure proper drying of the spin column membrane after washing to prevent ethanol carryover. Ensure Buffer RPE is at room temperature for elution. For very small RNA amounts, consider specialized RT kits.

Appendix A: Deparaffinization Methods

Paraffin removal is essential for proteinase K access. Recommended methods include:

• Heptane and Methanol: A two-step process involving heptane incubation, methanol wash, and air drying.
• Xylene, Limonene or CitriSolv: Dissolves paraffin, followed by ethanol wash and air drying.
• Melting: Melting paraffin to pierce and access the sample.

All methods involve subsequent steps similar to the main protocol, often using specific volumes indicated by ▲ or ●.

Appendix B: General Remarks on Handling RNA

RNases are stable enzymes that can degrade RNA. Strict aseptic techniques are required:

• Use RNase-free plasticware and glassware.
• Wear gloves and change them frequently.
• Keep tubes closed when not in use.
• Use RNase-free water and solutions.
• For decontamination, use solutions like 0.1 M NaOH, 1 mM EDTA, or chloroform, followed by RNase-free water and ethanol.
• DEPC treatment can be used for water and solutions, but must be followed by autoclaving to remove traces of DEPC.

Appendix C: Storage, Quantification and Determination of Quality of RNA

Storage: Purified RNA can be stored at -30°C to -15°C or -70°C in RNase-free water for over a year without degradation.

Quantification: RNA concentration is determined by measuring absorbance at 260 nm (A260) using a spectrophotometer. An A260 of 1 unit corresponds to 44 µg/ml RNA at neutral pH. For small amounts, specialized systems like QIAxcel or Agilent Bioanalyzer are recommended.

Purity: The A260/A280 ratio estimates purity. A ratio of 1.9–2.1 in 10 mM Tris-Cl, pH 7.5, indicates pure RNA. Measurements should be done in a neutral pH buffer.

Ordering Information

For ordering information, visit www.qiagen.com/shop.

Document Revision History

January 2020: Updated text and ordering information for QIAcube Connect.

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Use of this product signifies agreement to terms including: use only with provided protocols and kit components; QIAGEN grants no license for use with non-kit components; kit components are licensed for one-time use only; QIAGEN disclaims other licenses; users agree not to permit prohibited acts; QIAGEN may enforce prohibitions in court.

For updated license terms, see www.qiagen.com.

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