RNA Extraction by Sample Type

Which RNA Extraction Kit from FFPE Samples is Right for You?

The recovery of quality RNA from FFPE specimens can be quite challenging. The fixation process causes cross-linkage between nucleic acids and proteins, and covalently modifies RNA by the addition of monomethyl groups to the bases. As a result, the molecules are rigid and susceptible to mechanical shearing, and the cross-links may compromise the use of RNA as a substrate for reverse transcription. Therefore, in order to utilize FFPE tissues as a source for gene expression analysis, a reliable method is required for extraction of RNA from the cross-linked matrix.

There are a number of factors that can impact the overall quality and yield of RNA isolated from FFPE tissues. Here are recommendations to address several key factors:

1. Upstream tissue procurement and tissue specimen preparation—if possible, tissues should be fixed within one hour of surgical resection. Extensive degradation of RNA can occur before completion of the fixation process. The optimal fixation time is 12–24 hours, using neutral-buffered formalin or paraformaldehyde. Fixed tissues should be thoroughly dehydrated prior to the embedding process.
2. Block storage—storage of blocks without cut faces, when possible, prevents ongoing damage from exposure to atmospheric oxygen, water, and other environmental factors such as light and infestation (fungus, insects, etc.).
3. Tissue type, size, and amount being used for RNA isolation—the recommended tissue thickness is 10–20 µm The number of sections used is determined by the tissue type (which impacts cell density) and surface area (recommended size: 50–300 mm2). Excess starting material can cause filter clogging, resulting in poor yield.
4. Excessive amount of paraffin used for embedding tissues—when possible, excess paraffin should be trimmed away prior to starting the purification protocol. For xylene-based purification methods, two xylene treatments at room temperature should be sufficient for complete deparaffinization. If desired, a more rigorous 37–55°C treatment can be performed for up to 30 minutes. After the xylene deparaffinization, it is crucial that the 100% ethanol is completely removed and the pellets are dry after the two 100% ethanol washes. The magnetic bead method employs novel chemistries to deal with the paraffin that limits input to 20 µm sections.

The MagMAX FFPE Nucleic Acid (NA) isolation kits are designed for rapid, efficient, and automatable isolation of total RNA and DNA from FFPE samples using Invitrogen MagMAX magnetic particles. Invitrogen MagMAX FFPE NA kits offer faster workflows and use less toxic reagents, without sacrificing quality in the process. These kits help deliver nucleic acid yield and purity comparable to the best-in-class RecoverAll filter-based system. The MagMAX FFPE NA kits outperform products with similar protocols, and achieve RNA and DNA yields that are comparable with longer, more hazardous, and lower-throughput protocols.

The RecoverAll Total Nucleic Acid Isolation Kit procedure requires about 45 minutes of hands-on time and can easily be completed in less than 1 day when isolating RNA. FFPE samples are deparaffinized using a series of xylene and ethanol washes. Next, they are subjected to a rigorous protease digestion with an incubation time tailored for recovery of either RNA or DNA. The nucleic acids are purified using a rapid glass-filter methodology that includes an on-filter nuclease treatment, and are eluted into either water or the low-salt buffer provided.

TechNotes Articles

• Nucleic Acid Extraction from FFPE Samples
• Accurate, Sensitive Quantitation of microRNAs from FFPE Tissues - TechNotes 16(1): This study describes the successful isolation and accurate measurement of miRNA expression from formalin- or paraformalin-fixed, paraffin-embedded (FFPE) samples.
• Faster Nucleic Acid Isolation From FFPE Tissues - TechNotes 16(1): Applied Biosystems has improved the protocol for the Invitrogen Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE Tissues, resulting in significantly reduced processing time.
• Optimized Gene Expression Analysis from FFPE Samples - TechNotes 14(3): The optimized protocol presented here can be used to reliably quantify mRNA expression levels using RNA isolated from FFPE samples and real-time RT-PCR.
• Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue - TechNotes 12(3): Ambion's new RecoverAll Kit enables researchers to isolate DNA and/or RNA (including microRNA) from FFPE samples. The nucleic acids recovered are ideal for various downstream applications such as microarray analysis, qRT-PCR, and mutation screening.
• MicroRNA Analysis Using RNA Extracted from Matched Formalin-Fixed Paraffin-Embedded (FFPE) and Fresh Frozen Samples
• High correlation of miRNA quantitation data from matched FFPE and snap-frozen tissues using TaqMan MicroRNA Assays 
• DNA Genotyping from Human FFPE Samples - Reliable and Reproducible: Application Note  
• Optimized Extraction and Quantification of RNA from FFPE Samples for Gene Expression Analyses  
• Protocol: FFPE Small RNA Library Preparation for SOLiD Sequencing
• RecoverAll™ Total Nucleic Acid Isolation Kit

Tips from the Bench

• The Optimal Thickness of FFPE Sections for miRNA Isolation: Our scientists examine the effect of section thickness (5–40 µm) on miRNA isolation from FFPE tissues.
  

Your Data

• Identifying Cancer-Related mRNA Signatures in FFPE Patient Samples: The Yeatman laboratory at the H. Lee Moffitt Cancer Center and Research Institute (Tampa, FL) investigates the gene expression signatures that define colon, breast, and other cancers.
• LCM Staining Effects on Real-Time RT-PCR: Research is described demonstrating that the Ambion LCM Staining Kit can be successfully used on formalin-fixed and paraffin-embedded (FFPE) tissues without compromising either the yield or the quality of RNA extracted.

Application Note

• Optimized extraction and quantification of RNA from FFPE samples for gene expression analyses

Product Bulletin

• RecoverAll and MagMAX kits for nucleic acid isolation: Methylation analysis using FFPE samples