miRNeasy FFPE Kit

ホルマリン固定パラフィン包埋した組織切片からのmicroRNAおよびトータルRNA精製

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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

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miRNeasy FFPE Kitは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ オンライン注文による24時間年中無休の自動処理システム

✓ 知識豊富で専門的な製品&テクニカルサポート

✓ 迅速で信頼性の高い（再）注文

特徴

miRNA とトータル RNA の効率的な精製
ホルマリンによるクロスリンクを切断する新しい手法
RNA分解がなく効率的にRNAを遊離
わずか85 分でRNA が得られる能率化されたプロトコール

製品詳細

miRNeasy FFPE Kit は、ホルマリン固定パラフィン包埋（FFPE）組織切片から約18 ヌクレオチド（nt）以上のRNA を含むトータルRNA を精製します。本キットはリアルタイム定量RT-PCR のようなアプリケーションに利用できるmiRNA やその他のsmall RNA を含むRNA フラグメントを精製します。

パフォーマンス

miRNeasy FFPE Kitを用いたトータルRNAおよびmiRNAの収量と性能は、フェノール・クロロホルム抽出法や、他社の類似のキットを使用するmiRNA 精製法より優れています（図“”、”および“”）。

図参照

Effective real-time RT-PCR quantification.

Efficient RNA purification from FFPE tissues.

原理

ホルマリン固定された組織では、ダウンストリームアプリケーションでRNA の性能を阻害するRNA・RNA およびRNA・タンパク質間のクロスリンキングが生じます。miRNeasy FFPE Kit は、特別な溶解およびインキュベーション条件によりRNA のホルマリンによるクロスリンキングを元に戻します。特別に開発された溶解バッファーは、組織切片からRNA を効率的に遊離し、さらにRNA 分解を防ぎます。最適な結合条件では、約18 ヌクレオチド以上のトータルRNA の精製を実現します。

操作手順

miRNeasy FFPE の全ての操作は、わずか85 分で完了します。至適化された溶解バッファーはRNA 収量を低減することなく、わずか15 分のProteinase K 分解でサンプル溶解が可能です。溶解後、サンプルを80 ℃で15 分間インキュベートし、ホルマリンによるクロスリンキングを元に戻します。その後、 DNase Booster Bufferとの至適化されたDNaseステップでゲノムDNAを迅速に除去し、RNeasy MinElute Spin Column を用いて高濃度なRNA を精製します（フローチャート“”）。RNA をわずか14 ～ 30 μl で溶出するため、より少量の反応容量でダウンストリームアプリケーションを行なえます。

図参照

miRNeasy FFPE procedure.

アプリケーション

miRNeasy FFPE Kit は、リアルタイム定量RT-PCR のような様々なアプリケーションで使用できるトータルRNA を含むmiRNA を精製します。

裏付けデータと数値

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
ApplicationsJA	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

リソース

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

Download Safety Data Sheets for QIAGEN product components.

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

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 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