RT2 RNA QC PCR Arrays

PCRアレイによる解析を行なう前のRNA品質評価

Products

RT2 RNA QC PCR Arrays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。
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RT2 RNA QC PCR Array

Cat. No. / ID:  330291

RT2 Profiler QC Array
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Features

  • RNA分解度、阻害物質、DNAコンタミをテスト
  • ハイスループットでのRNA品質コントロールに最適
  • 時間を短縮し、試薬の無駄な消費を回避

Product Details

RT² RNA QC PCR Arrayは、既製あるいはカスタムRT2 Profiler PCR Arrayで解析を行なう前に、ヒト、マウス、ラット、イヌ、アカゲザルのRNAサンプルを品質評価するために設計されています。アレイには、RNA分解度、逆転写とPCR増幅の阻害物質、ゲノムDNAと一般的なDNAのコンタミをテストするためのPCRコントロールが入っています。

Principle

RT2 RNA QC PCR Array(ヒト、マウス、ラット、イヌ、アカゲザル用)は既製またはカスタムRT2 Profiler PCR Arrayで解析を実施する前に、RNAサンプルの品質を評価するために設計されています。アレイには、RNA分解度、逆転写とPCR増幅の阻害物質、ゲノムDNAと一般的なDNAのコンタミをテストするためのPCRコントロールが入っています(下記参照)。これらのコントロールのいずれかで良好な結果が得られない場合は、RNAがSYBR® GreenベースのリアルタイムPCR実験で偽陽性あるいは偽陰性結果を引き起こす可能性があります。

RT2 RNA QC PCR ArrayはリアルタイムPCR結果の信頼性を高めます。1枚のプレートで最大12サンプルまでのRNAの品質管理を同時に実行することが可能な本製品は、全研究にわたり多数のサンプルの整合性を確実にするのに特に有用です。RNAサンプルの品質テストにRT2 RNA QC PCR Arrayを使用することにより、品質の低いサンプルへのマスターミックスとPCRアレイの無駄な消費を回避することができます。

コントロール

RT² RNA QC PCR Arrayには以下のようなコントロールが入っています

  • RNA分解度:2種類のハウスキーピング遺伝子(HK1、HK2)
  • ゲノムDNAのコンタミ:Genomic DNA control (GDC)
  • ゲノムDNAのコンタミ:No reverse transcription control (NRT)
  • 一般的な非特異的DNAコンタミ:No-template control (NTC)
  • 逆転写反応阻害:Reverse transcription control (RTC)
  • PCR増幅阻害:Positive PCR control (PPC)

RT² RNA QC PCR Arrayのコントロールエレメントすべてを正確に解析するためには、RT² First Strand Kitを用いて、トータルRNAからcDNAテンプレートを合成する必要があります。

フォーマット

ヒト、マウス、ラット、イヌ、アカゲザル用のRT2 RNA QC PCR Arrayを以下のフォーマットからお選びいただけます。

  • RT2 RNA QC PCR Array Format A:96-well RT2 RNA QC PCR Array, 12 Optical Thin-Wall 8-Cap Strips
  • RT2 RNA QC PCR Array Format C:96-well RT2 RNA QC PCR Array, Optical Adhesive Film
  • RT2 RNA QC PCR Array Format D:96-well RT2 RNA QC PCR Array, 12 Optical Thin-Wall 8-Cap Strips
  • RT2 RNA QC PCR Array Format E:384-well RT2 RNA QC PCR Array, Optical Adhesive Film, 384EZLoad Covers
  • RT2 RNA QC PCR Array Format F:96-well RT2 RNA QC PCR Array, Optical Adhesive Film
  • RT2 RNA QC PCR Array Format G:384-well RT2 RNA QC PCR Array, Optical Adhesive Film, 384EZLoad Covers
  • RT2 RNA QC PCR Array Format R:Rotor-Disc 100 RT2 RNA QC PCR Array, Rotor-Disc Heat Sealing Film


Procedure

各cDNAテンプレート(RT2 First Strand Kitを用いて作製)とPCRマスターミックスを、HK1、HK2、GDC、RTC、および二つのPPCウエルの一つに、同じ容量で混和します。各RNAサンプルをPCRマスターミックスと混和し、サンプルのNRTウエルに添加します。リアルタイムPCRサイクリングプログラムを実行する前に、希釈したPCRマスターミックスをNTCおよびPPCのもう一つのウェルに添加します。

Applications

ヒト、マウス、ラット、イヌ、アカゲザル用のRT² RNA QC PCR Arrayは、RT2 Profiler PCR Arrayを用いたリアルタイムPCRベースの遺伝子発現解析を行なう前に、RNAサンプルの品質を評価するのに適しています。

Resources

パンフレット (1)
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
ダウンロードファイル (2)
Data analysis file for RT² Profiler PCR Array Housekeeping Genes
Catalog number- 330231
Pathway number- PAXX-000
RNA QC Data Analysis
XLS (484KB)

Data analysis file for RT² ProfilerRT² Profiler™ PCR Array RT2 RNA QC
Catalog number- 330231
Pathway number- PAXX-999

Safety Data Sheets (1)
Certificates of Analysis (1)
Brochures & Guides (1)
Download Files (2)
Data analysis file for RT² Profiler PCR Array Housekeeping Genes
Catalog number- 330231
Pathway number- PAXX-000
RNA QC Data Analysis
XLS (484KB)

Data analysis file for RT² ProfilerRT² Profiler™ PCR Array RT2 RNA QC
Catalog number- 330231
Pathway number- PAXX-999

FAQ

How can I ensure that reaction volume is not lost due to evaporation during thermal cycling?
Be sure to carefully and completely seal the qPCR assay plate with fresh, optical, thin-wall, 8-cap strips or adhesive optical film before the plate is placed into the real-time cycler. In addition, refer to your instrument's user's manual to determine whether the real-time cycler manufacturer recommends use of a plate compression pad during the run.
FAQ ID -2679
How do I create a workspace that is free of DNA contamination, prior to carrying out a qPCR experiment?

Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false-positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:

 

  • Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
  • Use only fresh PCR-grade reagents and disposable labware.
  • Treat any labware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
  • Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, and cloning.
  • Change the lab bench pads/papers often and decontaminate lab benches and labware (racks, pipettors, etc.) before each use by washing with 10% bleach, and/or exposing to UV light for at least 10 minutes. This serves to degrade and/or inactivate contaminating DNA.
  • Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.  
FAQ ID -2654
What testing should be performed to assess the quality of an RNA sample?

All RNA samples should be assessed spectrophotometrically (diluted in 10mM Tris, pH 8.0), and electrophoretically, and should meet the following specifications:

  • Total RNA concentration by A260 should be greater than 40 µg/ml
  • A260: A280 ratio should be 1.8 to 2.0
  • A260: A230 ratio should be greater than 1.7
  • Analysis of ~100ng of total RNA on an Agilent Bioanalyzer using an RNA 6000 Nano LabChip, or analysis of 1.5 μg of total RNA on a denaturing 2.0% agarose gel containing ethidium bromide (0.5 μg/ml) should contain sharp 28S and 18S rRNA bands, with no smearing at their low molecular weight edge. The 28S:18S band intensity ratio should be ~2:1. When utilizing the RNA 6000 Nano LabChip for RNA analysis, the RNA should have a RIN (RNA integrity) score of 7.0 or higher.

In addition to the above quality control tests, the RT2 RNA QC PCR Array for human (PAHS-999), mouse (PAMM-999), or rat (PARN-999) can be used. These arrays allow the rapid assessment of high and low housekeeping gene expression levels, reverse transcription and polymerase chain reaction efficiency, and genomic and general DNA contamination.

FAQ ID -2660
What is a dissociation curve, and why is it important to run a dissociation curve, following qPCR using SYBR Green chemistry?

Dissociation curves are carried out at the end of a PCR experiment by following a 3-step procedure.

First, all the components are denatured at 95°C, followed by complete annealing at a set temperature (based on the primer Tm values), followed by a gradual increase in temperature up to 95°C. Fluorescence intensity is monitored during this final temperature increase, resulting in the generation of a melting curve or dissociation curve.

By analyzing the first derivative of such a curve, you can readily assess the homogeneity of the PCR products, including the presence of primer–dimers, thereby determining the specificity of the PCR reaction. It is important to carry out such post-PCR analyses when using SYBR Green probe chemistry due to this reagent's lack of sequence specificity.

FAQ ID -2678