miRNeasy Tissue/Cells Advanced Kits

少量のサンプルを含む組織や細胞からのmicro RNAおよびトータルRNAをフェノールフリーで精製

S_1058_4_miRNeasyTissueCellsAdvancedMicro

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miRNeasy Tissue/Cells Advanced Micro Kit (50)

Cat. No. / ID:  217684

For 50 preps: RNeasy UCP MinElute spin columns, gDNA Eliminator Spin Columns, Collection Tubes, RNase-Free Water and Buffers
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$520.00
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Column typePlate type
Micro
Mini
96 well
miRNeasy Tissue/Cells Advanced Kits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。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • フェノールフリーなプロトコールのため相分離やドラフトでの作業が不要
  • 特長的なgDNA Eliminator Columnにより迅速で効率的な手順
  • miRNAおよび200塩基未満のRNAを効率的に濃縮
  • すべてのダウンストリーム解析に適したRNA精製

Product Details

miRNeasy Tissue/Cells Advanced Kitは、定評のあるmiRNeasy Kitの第二世代です。より安全に、そして効率的にRNA精製をするために、フェノールフリーの技術とgDNA Eliminator Columnを組み合わせたものです。少量サンプルを含む、動物組織や細胞から18塩基以上のスモールRNAを含むトータルRNAの精製を可能にします。

miRNeasy Tissue/Cells Advanced Kitは、ロースループットのRNA精製ではスピンカラムを使用し、ハイスループットの精製ではQIAcube Connectで自動化できます。

Performance

miRNeasy Tissue/Cells Advanced Kitは、従来のmiRNeasy Kitに代わるフェノールフリーキットで、RNAの収量と品質に妥協はありません。(図「 フェノール不要で高収量miRNA」および「 miRNeasy Tissue/Cells Advanced Microを使用してフェノール不要で組織や細胞からmiR16の高収量」を参照)。miRNeasy Tissue/Cells Advanced Kitは、およそ18塩基までのRNAを効率良く濃縮できます。
miRNeasy Tissue/Cells Advanced Micro Kitは、最大5 mgの凍結組織または1 x 106個の細胞からRNAを効率良く精製しますが、サンプルが少量の場合でも精製することができます。miRNeasy Tissue/Cells Advanced Mini Kitは、最大30 mgの凍結組織または1 x 107個の細胞からRNAを効率良く精製します。
miRNeasy Tissue/Cells Advanced Kitには、高感度なアプリケーション用にゲノムDNAの効率良い除去、かつ高収量で再現性の高いRNA精製を可能にするgDNA Eliminatorの手順が含まれます(図「 miRNeasy Tissue/Cells Advanced Mini Kitでの高パフォーマンス」および「 トータルRNAの精製:ゲノムDNAの効果的な除去」を参照)。通常、組織や培養細胞からAgilent RIN値が10に近いトータルRNAが得られます。他社キットとは異なり、miRNeasy Tissue/Cells Advanced Mini Kitは、RNAの品質と収量を妥協することなく、使いやすさとフェノールフリーのプロトコールを組み合わせています(図「 miRNeasy Tissue/Cells Advanced Mini Kitは、他社キットよりも優れたパフォーマンスを示す」、「 優位性のあるパフォーマンス」、「 迅速な手順」を参照)。

See figures

Principle

miRNeasy Tissue/Cells Advanced Kitは、グアニジンベースのサンプル溶解と、阻害剤除去のステップやシリカメンブレンベースのトータルRNA精製(miRNAとその他スモールRNAを含む)を組み合わせています。キットに同梱のBuffer RLTは、RNaseなどのタンパク質の溶解と変性を促進するグアニジンチオシアン酸塩を含んでいます。そのため、Buffer RLTで溶解したサンプル内のRNAは安定的で、分解から保護されます。

Procedure

サンプルの完全な溶解とRNaseの不活化を確実に行うために、組織や細胞サンプルにBuffer RLTを添加し、続いて徹底した破砕とホモジナイゼーションを行います(図「 miRNeasy Tissue/Cells Advanced Kitの手順」を参照)。 次に、Buffer ALを溶解液に添加し、gDNA Eliminator Spin ColumnでgDNAの除去を行います。サンプルの種類や量によりますが、カラム上でDNase処理のオプションを実行できます(本キットには含まれていません)。Buffer RPPをフロースルーに添加し、阻害物質(主に組織や細胞サンプル内に高濃縮に存在し、RNA分離やダウンストリームの解析に影響を与える可能性のあるタンパク質)を遠心分離で沈殿させます。トータルRNAを含む上清を新しい反応チューブに移し、18塩基以上のすべてのRNA分子に適した結合条件にするため、イソプロパノールを添加します。その後、サンプルをRNeasy Mini Spin Column(miRNeasy Tissue/Cells Advanced Mini)またはRNeasy UCP MinElute Spin Column(miRNeasy Tissue/Cells Advanced Micro)に添加し、トータルRNAをメンブレンに結合させ、夾雑物はすべて効率的に洗浄されます。miRNAやその他のスモールRNAを含む高品質なRNAは少量のRNase-Free Waterで溶出します。

See figures

Applications

miRNeasy Tissue/Cells Advanced Kitは、以下を含むさまざまなアプリケーション用のmiRNAやトータルRNAの精製を可能にします。

  • RNAシーケンス
  • 定量、リアルタイムRT-PCR
  • マイクロアレイ解析
  • ノーザンブロット解析

機能 miRNeasy Tissue/Cells Advanced Micro miRNeasy Tissue/Cells Advanced Mini
アプリケーション NGS、リアルタイムRT-PCR、マイクロアレイ NGS、リアルタイムRT-PCR、マイクロアレイ
溶出量 既定値 20 µl, 最小値 10 µl 30 µl
フォーマット スピンカラム スピンカラム
主なサンプルの種類 組織、細胞 組織、細胞
処理 手動(遠心分離)、自動(QIAcube Connect) 手動(遠心分離)、自動(QIAcube Connect)

精製産物

miRNA、トータルRNA miRNA、トータルRNA
サンプル量 最大 5 mg の組織、最大 1 x 106 個の細胞

最大 30 mg の凍結組織(15 mg 安定化)、

最大 1 x 107 個の細胞

Supporting data and figures

Resources

キットハンドブック (3)
クイックスタートプロトコール (3)
アプリケーション/プロトコール (1)
Certificates of Analysis (1)
Quick-Start Protocols (3)
Kit Handbooks (3)

Publications

Accurate isoform discovery with IsoQuant using long reads.
Prjibelski AD; Mikheenko A; Joglekar A; Smetanin A; Jarroux J; Lapidus AL; Tilgner HU;
Nat Biotechnol; 2023; 41 (7):915-918 2023 Jan 2 PMID:36593406
Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity.
Chryplewicz A; Scotton J; Tichet M; Zomer A; Shchors K; Joyce JA; Homicsko K; Hanahan D;
Cancer Cell; 2022; 40 (10):1111-1127.e9 2022 Sep 15 PMID:36113478
Developmental defects in Huntington's disease show that axonal growth and microtubule reorganization require NUMA1.
Capizzi M; Carpentier R; Denarier E; Adrait A; Kassem R; Mapelli M; Couté Y; Humbert S;
Neuron; 2021; 110 (1):36-50.e5 2021 Nov 17 PMID:34793694
T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles.
Céspedes PF; Jainarayanan A; Fernández-Messina L; Valvo S; Saliba DG; Kurz E; Kvalvaag A; Chen L; Ganskow C; Colin-York H; Fritzsche M; Peng Y; Dong T; Johnson E; Siller-Farfán JA; Dushek O; Sezgin E; Peacock B; Law A; Aubert D; Engledow S; Attar M; Hester S; Fischer R; Sánchez-Madrid F; Dustin ML;
Nat Commun; 2022; 13 (1):3460 2022 Jun 16 PMID:35710644
Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients.
Giannella A; Riccetti S; Sinigaglia A; Piubelli C; Razzaboni E; Di Battista P; Agostini M; Dal Molin E; Manganelli R; Gobbi F; Ceolotto G; Barzon L;
Front Immunol; 2022; 13 :968991 2022 Aug 11 PMID:36032130

FAQ

How much RNA does a typical mammalian cell contain?

The RNA content and RNA make up of a cell depend very much on its developmental stage and the type of cell. To estimate the approximate yield of RNA that can be expected from your starting material, we usually calculate that a typical mammalian cell contains 10–30 pg total RNA.

The majority of RNA molecules are tRNAs and rRNAs. mRNA accounts for only 1–5% of the total cellular RNA although the actual amount depends on the cell type and physiological state. Approximately 360,000 mRNA molecules are present in a single mammalian cell, made up of approximately 12,000 different transcripts with a typical length of around 2 kb. Some mRNAs comprise 3% of the mRNA pool whereas others account for less than 0.1%. These rare or low-abundance mRNAs may have a copy number of only 5–15 molecules per cell.

FAQ ID -2946
How can I check the integrity of RNA purified using RNeasy Kits?

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
What can I use to isolate RNA smaller than 200 nucleotides?

For the isolation of microRNA (miRNA) specifically, we have developed the miRNeasy Mini Kit and the miRNeasy 96 Kit for isolation from cells and tissues.   We also have the PAXgene Blood miRNA and Tissue miRNA kits for isolation from blood stored in PAXgene Blood RNA tubes and PAXgene Tissue Containers, respectively.  Other miRNA isolation supplementary protocols can also be found by searching our comprehensive protocols at http://www.qiagen.com/literature/default.aspx?WT.svl=m.

FAQ ID -115
What is the composition of Buffer RWT?
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
Why is the protein precipitation step optional when working with cells?
Cells do not contain as much proteins as tissue does, therefore a removal of proteins before RNA isolation is not necessary.
FAQ ID-3752
How do I clean up RNA preparations containing miRNA?

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
Do I have to discard Buffer RLT with beta-Mercaptoethanol (ß-ME) added to it after 1 month of storage?
No. Beta-Mercaptoethanol (ß-ME) is stable for 1 month, but Buffer RLT itself is stable for at least 9 months at room temperature (15 to 25°C). Simply add fresh ß-ME to the Buffer RLT supplied in RNeasy Kits to ensure complete inactivation of RNases while isolating RNA.
FAQ ID -1037
What are the effects of low A260/A230 ratios in RNA preparations on downstream applications?

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
What is the composition of Buffer RPE?
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
Can I use total RNA for the miRNA PCR Arrays or Assays?
Yes, you can. In fact, total RNA is the recommended starting material for the miScript PCR System. We recommend using the miRNeasy Mini Kit (217004) to isolate total RNA for use with the miScript PCR System.
FAQ ID -2726
Why do I have to add Buffer AL before using the gDNA Eliminator?
This is to optimize gDNA removal. 
 
FAQ ID-3751