miRNeasy FFPE Kit

For purification of microRNA and total RNA from formalin-fixed, paraffin-embedded tissue sections

Features

Effective purification of miRNA and total RNA
Novel method to overcome formalin crosslinking
Efficient release of RNA without compromising integrity
Streamlined protocol providing RNA in just 85 minutes

Useful Resources

miRNeasy FFPE Handbook miRNeasy FFPE Kit Quick Start Protocol Part 1 (EN) miRNeasy FFPE Kit Quick Start Protocol Part 2 (EN)

Efficient RNA purification from FFPE tissues.

Effective real-time RT-PCR quantification.

miRNeasy FFPE Kit (50)

Cat. No. / ID: 217504

50 RNeasy MinElute Spin Columns, Collection Tubes, Proteinase K, RNase-Free DNase I, DNase Booster Buffer, RNase-Free Buffers, RNase-Free Water

The miRNeasy FFPE Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The miRNeasy FFPE Kit enables purification of total RNA that includes RNA from approximately 18 nucleotides upwards from formalin-fixed, paraffin-embedded (FFPE) tissue sections. The kit provides recovery of usable RNA fragments, including miRNA and other small RNAs, for applications such as quantitative, real-time RT-PCR. Procedures can be automated on the QIAcube Connect.

Performance

Yields and performance of total RNA and miRNA with the miRNeasy FFPE Kit are superior to alternative methods of miRNA purification, such as using phenol-chloroform extraction or a similar kit from another supplier (see figures "", "" and ).

See figures

Effective real-time RT-PCR quantification.

Efficient RNA purification from FFPE tissues.

Superior yields and performance.

Principle

Fixing tissues with formalin leads to RNA-RNA and RNA-protein crosslinking which impairs RNA performance in downstream applications. The miRNeasy FFPE Kit provides special lysis and incubation conditions to reverse formalin crosslinking of RNA. A specially developed lysis buffer efficiently releases RNA from tissue sections while avoiding further RNA degradation. The lysate is treated with DNase then optimized binding conditions allow purification of all usable RNA from approximately 18 nucleotides upwards.

Procedure

The entire miRNeasy FFPE procedure can be completed in as little as 85 minutes. The optimized lysis buffer allows sample lysis with proteinase K digestion in only 15 minutes without sacrificing RNA yields. After lysis, samples are incubated at 80ºC for 15 minutes to reverse formalin crosslinking. Genomic DNA is then rapidly removed in an optimized DNase step with DNase Booster Buffer, and concentrated RNA is purified using RNeasy MinElute spin columns (see flowchart ""). Since RNA is eluted in a volume of just 14–30 µl, smaller reaction volumes are possible in downstream applications.

See figures

miRNeasy FFPE procedure.

Applications

The miRNeasy FFPE Kit allows purification of miRNA with total RNA for use in a variety of applications, such as quantitative, real-time RT-PCR.

Supporting data and figures

Efficient RNA purification from FFPE tissues.

Total RNA including miRNA was purified from the indicated rat tissues using either the miRNeasy FFPE Kit or phenol-chloroform extraction. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16 using the miScript PCR System. Results showed that for lung and liver tissues, C_T values were lower after purification using the miRNeasy FFPE Kit, indicating that higher amounts of miRNA were purified than when using phenol-chloroform extraction. For kidney tissue, C_T values were similar for both methods. C_T values were similar or lower after purification using the miRNeasy FFPE Kit for all tissue types tested.

Specifications

Features	Specifications
Applications	PCR, qPRC, real-time RT-PCR, microArray
Elution volume	14-30 µl
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein	miRNA, total RNA
Sample amount	up to 4 sections, each with a thickness up to 10 µm and a surface area up to 250mm^2 (automated protocol on QIAcube: up to 2 sections)
Processing	Manual (protocol for automated processing on QIAcube available)
Main sample type	FFPE tissue samples
Format	Spin column
Technology	Silica technology
Yield	Varies

Resources

Sample to Insight solutions for successful molecular analysis

High-quality, nucleic acid purification for successful PCR and NGS experiments.

Critical factors for molecular analysis of FFPE samples

Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow

For purification of total RNA, including miRNA, from formalin-fixed, paraffin-embedded tissue sections

Poster for download

FAQ

Paraffin-embedded or fixed samples typically yield fragmented, partially degraded RNA. In addition, RNA quality will depend greatly on the handling of the samples before, during, and after the fixation procedure.

If performing RT-PCR with degraded RNA, we recommend using gene-specific primers or random nonamers rather than oligo-dT primers, since the mRNA poly-A tail may have been lost due to degradation.

We recommend that the RNeasy FFPE or miRNeasy FFPE kit be used to isolate the RNA.

FAQ ID -828

The exact composition of Buffer PKD is proprietary. Buffer PKD functions as a Proteinase K Digestion Buffer and is a component of, for example, the AllPrep DNA/RNA FFPE Kit, RNeasy FFPE Kit, and the miRNeasy FFPE Kit. We are sometimes asked if Buffer PKD comprises any RNase inhibitors or RNase inhibiting agents — since the formalin-fixation of the starting material has already inactivated the RNases, no such reagents are present in this buffer.

FAQ ID -2801

I received one of the kits in the list below containing the MinElute columns, however they were left out for a while and not stored at 2-8°C upon receipt. Can I still use them? AllPrep DNA/RNA Micro, EpiTect Fast DNA Bisulfite, EpiTect Fast FFPE Bisulfite, EpiTect Fast LyseAll Bisulfite, EpiTect Plus DNA Bisulfite, EpiTect Plus FFPE Bisulfite, EpiTect Plus LyseAll Bisulfite, exoRNeasy Serum/plasma Maxi, exoRNeasy Serum/Plasma Midi, GeneRead DNA FFPE, GeneRead rRNA Depletion, GeneRead Size Selection, MinElute Gel Extraction, MinElute PCR Purification, MinElute Reaction Cleanup, miRNeasy FFPE, miRNeasy Micro, miRNeasy Serum/Plasma, QIAamp DNA FFPE, QIAamp DNA Investigator, QIAamp DNA Micro, QIAamp MinElute Media, QIAamp MinElute Virus Spin, QIAamp MinElute Virus Vacuum, RNeasy FFPE, RNeasy Micro, RNeasy Plus Micro

The MinElute spin columns should be stored at 2-8°C upon arrival. Short-term storage (up to 4 weeks) at room temperature (15-25°C) does not affect the performance. However, for optimal performance and quality, storage temperature should not exceed 25°C.

FAQ ID - 3560

The exact composition of Buffer RWT is confidential. Buffer RWT is a proprietary component of, for example, the miRNeasy Mini Kit and the RNeasy Plus Universal Kit. Guanidine salt and ethanol are important ingredients in Buffer RWT. Ethanol is added by the user prior to the first use of the kit. Buffer RWT is a stringent washing buffer used after preclearing the sample with QIAzol Lysis Reagent, especially if isolation of small RNAs, for example, microRNAs or RNAs from formalin-fixed tissue, is desired

FAQ ID -2798

RNA preparations containing miRNA can be cleaned up by modifying* the cleanup protocols listed in the handbooks of the RNeasy Mini Kit or the RNeasy MinElute Cleanup Kit .

* Modify the cleanup protocol at step 2, by increasing the volume of ethanol (96-100%) from 250 µl to 950 µl.

FAQ ID -3002

Unlike the Appendix A Protocol for the miRNeasy Mini Kit, which allows removal of larger RNAs such as mRNA and rRNA to enrich miRNA in a separate small RNA fraction, this is not practical using the miRNeasy FFPE Kit. RNA from FFPE- or other compromised materials is usually strongly fragmented already. Significant amounts of rRNA and mRNA fragments would end up in the small RNA-enriched fraction.

FAQ ID -1737

The exact composition of Buffer RPE is confidential. Buffer RPE is a mild washing buffer, and a proprietary component of RNeasy Kits. Its main function is to remove traces of salts, which are still on the column due to buffers used earlier in the protocol. Ethanol, which is added by the user just before using the kit for the first time, is an important ingredient of Buffer RPE.

FAQ ID -2797