RNeasy FFPE Kit for RNA Extraction

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

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Get in touch with our team today and request a quote for your RNeasy FFPE Kit (50) trial kit.

RNeasy FFPE Kit (50)

Cat. No. / ID: 73504

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

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

Want to try this solution for the first time?

Get in touch with our team today and request a quote for your RNeasy FFPE Kit (50) trial kit.

Request a quote for a trial kit

Features

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

Product Details

The RNeasy FFPE Kit is specially designed for purifying total RNA from formalin-fixed, paraffin-embedded tissue sections. Special lysis and incubation conditions reverse formaldehye modification of RNA. In addition, the lysis buffer efficiently releases RNA from tissue sections while avoiding further RNA degradation. The kit also uses DNase and DNase Booster Buffer for optimized removal of genomic DNA contamination. RNeasy MinElute spin columns enable purification of total RNA with elution volumes of as low as 10 μl. Purification can be fully automated on the QIAcube Connect.

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Performance

With the RNeasy FFPE Kit, yields of RNA purified from fixed and embedded samples are greater than those achieved using other methods (see figure “”). Recovered RNA is intact down to 70 nucleotides in length and virtually free of genomic DNA contamination (see figure “”). Usable RNA fragments purified with the kit from FFPE samples are highly suited for use in sensitive downstream applications, including real-time RT-PCR (see figures “” and “”).

See figures

Greater yields of RNA.

Recovery of all usable RNA.

Reliable results in real-time RT-PCR analysis.

Successful real-time RT-PCR analysis.

Principle

Fixing tissues with formalin leads to RNA–RNA and RNA–protein crosslinking which impairs RNA performance in enzymatic assays. Fixation and embedding conditions can also result in heavily fragmented nucleic acids in FFPE samples. The RNeasy FFPE Kit uses special lysis and incubation conditions to reverse formaldehyde modification of RNA. The lysis buffer efficiently releases RNA from FFPE tissue samples, preventing further RNA degradation. These optimized conditions allow purification of all usable RNA, leading to greater yields from FFPE samples with the RNeasy FFPE Kit than with other methods.

Procedure

The entire RNeasy FFPE procedure can be completed in as little as 85 minutes (see flowchart " RNeasy FFPE procedure"). Sample lysis with proteinase K digestion requires only 15 minutes. After lysis, samples are incubated at 80ºC for 15 minutes. DNase treatment then effectively removes genomic DNA, including small DNA fragments. Finally, concentrated RNA is purified using RNeasy MinElute spin columns, and eluted in a volume of 14–30 µl. Purification can be fully automated on the QIAcube.

See figures

RNeasy_FFPE_Procedure

Applications

The RNeasy FFPE Kit isolates all RNA molecules longer than 70 nucleotides from FFPE samples, providing usable RNA fragments for numerous downstream applications, including RT-PCR. However, RNA purified from FFPE samples is heavily fragmented and should not be used in downstream applications that require full-length RNA. Some applications may require modifications to allow the use of fragmented RNA (e.g., designing small amplicons for RT-PCR). For cDNA synthesis, either random or gene-specific primers should be used instead of oligo-dT primers.

Supporting data and figures

Reliable results in real-time RT-PCR analysis.

Total RNA was purified from a 10 µm rat lung FFPE sample using the RNeasy FFPE Kit or alternative kits from Supplier E_III, Supplier N, Supplier A_IV, and Supplier M. Real-time RT-PCR assays for PGK1 (phosphoglycerate kinase 1) were performed on the Rotor-Gene Q cycler with (+RT) or without (-RT) the reverse transcription step. The high C_T value in the -RT sample from the RNeasy FFPE Kit indicates that purified RNA is virtually free of genomic DNA. Therefore the C_T value in the +RT sample from the RNeasy FFPE Kit is a reliable measure of RNA expression.

Specifications

Features	Specifications
Applications	PCR, qPCR, real-time RT-PCR, microarray
Elution volume	14–30 µl
Main sample type	FFPE tissue samples
Processing	Manual (centrifugation), automated (QIAcube)
Format	Spin column
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein	RNA
Time per run or per prep	70 minutes
Sample amount	15 µm to 410µm sections
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.

FFPE サンプルの分子解析における重要なファクター

Critical factors for molecular analysis of FFPE samples

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

Publications

Determinants of RNA quality from FFPE samples.

von Ahlfen S; Missel A; Bendrat K; Schlumpberger M;

PLoS One; 2007; 2 (12):e1261 2007 Dec 5 PMID:18060057

FAQ

Yes, we offer the RNeasy FFPE Kit specially designed to purify total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. It provides special lysis and incubation conditions to reverse formaldehyde modification of RNA typical for formalin fixed tissues.

FAQ ID -1174

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

The MinElute spin columns included in the following kits should be stored at 2–8°C upon arrival: 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.

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 RNeasy 96 Universal Tissue Kit enables high-throughput purification of RNA from any animal or human tissue sample, including difficult-to-lyse fibrous and fatty tissues. For single tube format RNA purification, the RNeasy Plus Universal Kit is also available.

Please refer to the Selection guide for RNA isolation for all sample types to find the optimal solution for your sample source.

FAQ ID -627

The exact composition of Buffer RLT is confidential. This buffer is a proprietary component of RNeasy Kits. Buffer RLT contains a high concentration of guanidine isothiocycanate, which supports the binding of RNA to the silica membrane. Buffer RLT can be purchased separately (cat. no. 79216)

Note: note that ß-mercaptoethanol should be added to Buffer RLT before use to effectively inactivate RNAses in the lysate (10 µl ß-Mercaptoethanol per 1 ml Buffer RLT).

FAQ ID -2793

The integrity and size distribution of total RNA purified with RNeasy Kits can be checked by denaturing-agarose gel electrophoresis, the Agilent 2100 bioanalyzer, or the QIAxcel Advanced System with the QIAxcel RNA QC Kit v2.0.

The respective ribosomal species should appear as sharp bands on the stained gel. 28S ribosomal RNA bands should be present with an intensity approximately twice that of the 18S RNA band. If the ribosomal bands are not sharp, but appear as a smear of smaller sized RNAs, it is likely that the RNA sample has suffered major degradation during preparation.

Size of ribosomal RNAs from various sources

Source	rRNA	Size (kb)
E. coli	16S	1.5
	23S	2.9
S. cerevisiae	18S	2.0
	26S	3.8
Mouse	18S	1.9
	28S	4.7
Human	18S	1.9
	28S	5.0

FAQ ID -1024

A maximum of 5 µl RNA eluate from RNeasy extraction procedures can be added to the reverse-transcription reaction with the QuantiTect Whole Transcriptome Kit.

FAQ ID -1616

Small amounts of RNA and DNA may be difficult to measure spectrophotometrically. Fluorometric measurements, or quantitative RT-PCR and PCR are more sensitive and accurate methods to quantify low amounts of RNA or DNA.

Fluorometric measurements are carried out using nucleic acid binding dyes, such as RiboGreen® RNA Quantitation Reagent for RNA, and PicoGreen® DNA Quantitation Reagent for DNA (Molecular Probes, Inc.).

FAQ ID -728

Tissue-Tek O.C.T. is an embedding compound for cryosectioning, which is soluble in water. It mainly consists of glycols and synthetic resins. Tissue-Tek O.C.T. is used as matrix for cryosectioning of tissues. Using the O.C.T. the tissue samples can be positioned more easily in the microtome and have better qualities during sectioning.

Most cryosections are fixed using non-crosslinking agents. For isolation of RNA from tissue embedded in Tissue-Tek O.C.T. using non-crosslinking agents the RNeasy Plus Micro Kit or the RNeasy Micro Kit (Protocol: Total RNA Isolation from Microdissected Cryosections), or the RNeasy Mini Kit (RNeasy Mini Protocol for the isolation of Total RNA from Animal Tissue) give great results. We strongly recommend removing as much of the embedding compound as possible prior to RNA extraction from the sections. Please see QIAGEN News article, Issue 1 1998, 'Effects of malnutrition on expression of lactase in children' for successful RNA isolation from O.C.T.-embedded tissue using the RNeasy Mini Kit.

In case crosslinking agents (e.g. formaldehyde or glyoxal-containing) were used for fixation of the tissue for cryosectioning the RNeasy FFPE Kit is the perfect choice. The RNeasy FFPE Kit is especially designed for purifying total RNA from formalin-fixed tissue sections. Special lysis and incubation conditions reverse formaldehyde modification of RNA for improved results in downstream application. The crosslinking causes the RNA to break, resulting in overall smaller molecules, which look like a smear when analyzed on a formaldehyde gel.

In case DNA as well as RNA from precious samples is of interest from the identical sections, please have a look at the AllPrep DNA/RNA Kits for unfixed tissue or the AllPrep DNA/RNA FFPE Kit for fixed tissue.

FAQ ID -530

The efficiency of downstream applications depends strongly on the purity of the RNA sample used. Pure RNA should yield an A260/A230 ratio of around 2 or slightly above; however, there is no consensus on the acceptable lower limit of this ratio. Possible candidates that can increase the A230 include “salt”, carbohydrates, peptides, and phenol (or aromatic compounds in general). In our experience, the increased absorbance at 230 nm in RNA samples is almost always due to contamination with guanidine thiocyanate, present at very high concentrations in the lysis buffer or extraction reagent used in most RNA purification procedures.

Please find an article discussing the effect of low 260/230 ratios in RNA preparations on downstream applications on page 7 of QIAGEN Newsletter March 15, 2010 . In summary, we found that concentrations of guanidine thiocyanate of up to 100 mM in an RNA sample do not compromise the reliability of downstream applications.

FAQ ID -2248

To ensure efficient gDNA removal when doing an on-column digest using the RNase-Free DNase Set in combination with RNeasy Mini the following factors are crucial:

prevent overloading by adjusting the amount of starting material to no more than the maximum amounts recommended in the RNeasy Mini Handbook
ensure complete disruption and homogenization of the starting material as instructed in the section 'Disruption and homogenization of starting materials' of the handbook
strictly follow the protocol for on-column DNase Digestion in Appendix D of the RNeasy Mini Handbook (you can let wash buffer RW1 incubate on the column for 3-5 minutes before centrifuging to enhance removal of excess gDNA prior to applying the enzyme)

In the rare case that trace amounts of genomic DNA are still detectable in sensitive downstream applications such as e.g., realtime RT-PCR, an in-solution digest using the RNase-Free DNAase set can be performed. Instructions are presented in Appendix C of the RNeasy MinElute Cleanup Handbook.

Alternatively, a second on-column digest can be carried out in future preparations, immediately following the RW1 wash after the first incubation with DNase.

FAQ ID -1087

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

The exact composition of Buffer RW1 is confidential. Buffer RW1 is a proprietary component of RNeasy Kits. Buffer RW1 contains a guanidine salt, as well as ethanol, and is used as a stringent washing buffer that efficiently removes biomolecules such as carbohydrates, proteins, fatty acids etc., that are non-specifically bound to the silica membrane. At the same time, RNA molecules larger than 200 bases remain bound to the column.

Note: Buffer RW1 should not be used for isolation of small RNAs, for example, microRNAs or fragmented RNA from formalin-fixed tissues, as these smaller fragments will be washed away. Buffer RWT should be used instead.

FAQ ID -2796