RT2 Profiler PCR Arrays

信頼性と感度の高い遺伝子発現プロファイリング

Products

RT2 Profiler 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 Profiler PCR Array

Cat. No. / ID:  330231

RT2 Profiler PCR Array
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RT2 RNA QC PCR Array

Cat. No. / ID:  330291

RT2 Profiler QC Array
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Features

  • 遺伝子パネルの発現をプロファイル
  • 簡単なリアルタイムPCR法で高い感度
  • プレアンプ使用で、わずか1 ngのトータルRNAで使用可能
  • 一般的なリアルタイムPCR装置を使用可能
  • 無料のオンラインツールを用いて簡単なデータ解析

Product Details

RT2 Profiler PCR Arrayは、パスウェイあるいは疾病に特異的な遺伝子に焦点をあてたリアルタイムPCRプライマーアッセイセット(至適化済み)で、 適切なRNA品質コントロールも含まれており、96ウェルプレート、384ウェルプレート、100-well discフォーマットでお届けします。RT2 Profiler PCR Arraysは、リアルタイムPCRの感度と、複数の遺伝子のプロファイリングが可能なマイクロアレイの特長を持ち合わせた遺伝子発現解析を実現します。

Performance

感度

RT2 First Strand Kitの感度により、アレイプレートあたりわずか1 ngまたは最高5 µgのトータルRNAで80%を超えるpresent call rateが得られます(図“ わずか25 ngのRNAでポジティブな結果”)。

再現性

PCRアレイトータルシステムは、技術的なreplicate実験、異なるロット間、異なる装置間で高い相関性(相関係数が0.99を超える)をもつことが証明され、生体サンプルの発現の差を高い信頼性で検出できます(図“ 異なるユーザー間で高い再現性”)。

特異性

高品質のスタートRNAを用いたPCRアレイシステムでは、プライマーダイマーや二次産物がなく、予想されたサイズの単一バンドが得られるため、高精度なリアルタイムPCR 結果を実現します(図“ すべての反応で単一の遺伝子特異的産物”)。

一定したPCR増幅効率

全遺伝子および全サンプルを通じて遺伝子発現レベルの正確な比較を行なうためには、一定したPCR増幅効率がPCRアレイテクノロジーに必須です。当社独自のプライマー設計アルゴリズムと、全プライマーアッセイの厳密なテストにより、PCRアレイでの全プライマーアッセイの高いパフォーマンスを保証します(図“ PCRアレイは高精度な結果を実現”)。

See figures

Principle

RT2 Profiler PCR Arrayは、フォーカスした遺伝子パネルの発現解析のための信頼できるツールです。各96ウエルプレート、384ウエルプレート、100-well disc PCR Arrayは、徹底的に研究されたパスウエイや疾病に関連した遺伝子パネル用のプライマーアッセイ(SYBR® Greenに至適化済み)を提供します。RT2 Profiler PCR Arrayはまたお客様の研究目的に即した遺伝子パネルをご提供するためにカスタマイズも可能です。高品質なプライマー設計とRT2 SYBR® Green qPCR Mastermix成分により、PCR アレイは同一サイクリング条件下で、96あるいは384種類の遺伝子特異的産物の同時増幅を実現します。

この組み合わせにより、RT2 Profiler PCR Arrayで正確なリアルタイムSYBR® Green結果に必要な高い感度と増幅効率が得られます。PCRアレイはどのような研究室でも容易に使用できます。

RT2 Profiler PCR Arrayは通常のサンプル(0.1~5 µgのRNA)、 FFPEサンプル、少量のサンプル(1~100 ngのRNA)から調製したRNAを使用して十分な感度を発揮できます。

Procedure

cDNAテンプレートと適切で即使用可能なPCRマスターミックスを混和し、同一プレートの各ウエルに等量分注し、リアルタイムPCRサイクリングプリグラムを起動します(フローチャート“ 簡単な操作”)。RT2 Profiler PCR Arrayは、QIAGEN、ABI、Bio-Rad、Eppendorf、Roche、Stratageneの装置で使用可能です。

適応性が高いレイアウトとコントロール

RT2 Profiler PCR Arrayは96ウエルプレート、384ウエルプレート、100-well discフォーマットで入手可能で、84あるいは370種類の疾病あるいはパスウエイに関連する遺伝子と5種類のハウスキーピング遺伝子の発現を調べることができます。RT2 Profiler PCR Arrayには以下の目的のためにコントロールが入っています:

  • データの標準化  
  • ゲノムDNAのコンタミを検出
  • RNAサンプル品質 
  • 一般的なPCRパフォーマンス
使いやすいデータ解析

エクセルベースのデータ解析テンプレートあるいはウエブベースのソフトウェアを用いてデータ解析を簡単に行なえます。データ解析はΔΔCT法に基づき、ハウスキーピング遺伝子に対して生データを標準化します。

See figures

Applications

RT2 PCR Profiler Arrayは次のような生物および医学研究領域で使用できます。

  • がん
  • 炎症およびサイトカインのプロファイリング
  • 幹細胞
  • 神経化学
  • シグナル伝達パスウェイ
  • 細胞接着と細胞移動
  • バイオマーカーのスクリーニングと検証

Supporting data and figures

Resources

パンフレット (3)
Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
ダウンロードファイル (3)
For analyzing gene expression data from RT2 Profiler PCR Arrays 
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

サイエンティフィック・ポスター (1)
Poster for download
キットハンドブック (1)
For pathway-focused gene expression profiling using real-time RT-PCR
機器テクニカル資料 (2)
For gene expression and genomic analysis
For pathway-focused gene expression analysis
Certificates of Analysis (1)
Brochures & Guides (3)
Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow
Download Files (3)
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

For analyzing gene expression data from RT2 Profiler PCR Arrays 
Kit Handbooks (1)
For pathway-focused gene expression profiling using real-time RT-PCR
Instrument Technical Documents (2)
For pathway-focused gene expression analysis
For gene expression and genomic analysis
Scientific Posters (1)
Poster for download

Publications

CD8 T cells use IFN-γ to protect against the lethal effects of a respiratory poxvirus infection.
Goulding J; Abboud G; Tahiliani V; Desai P; Hutchinson TE; Salek-Ardakani S;
J Immunol; 2014; 192 (11):5415-25 2014 Apr 18 PMID:24748494
Sphingosine kinase 1 expressed by endothelial colony-forming cells has a critical role in their revascularization activity.
Poitevin S; Cussac D; Leroyer AS; Albinet V; Sarlon-Bartoli G; Guillet B; Hubert L; Andrieu-Abadie N; Couderc B; Parini A; Dignat-George F; Sabatier F;
Cardiovasc Res; 2014; 103 (1):121-30 2014 Apr 17 PMID:24743591
Adipose tissue insulin resistance due to loss of PI3K p110α leads to decreased energy expenditure and obesity.
Nelson VL; Jiang YP; Dickman KG; Ballou LM; Lin RZ;
Am J Physiol Endocrinol Metab; 2014; 306 (10):E1205-16 2014 Apr 1 PMID:24691033
FBXW7 mutations in melanoma and a new therapeutic paradigm.
Aydin IT; Melamed RD; Adams SJ; Castillo-Martin M; Demir A; Bryk D; Brunner G; Cordon-Cardo C; Osman I; Rabadan R; Celebi JT;
J Natl Cancer Inst; 2014; 106 (6):dju107 2014 May 16 PMID:24838835
β-Carotene-9',10'-oxygenase status modulates the impact of dietary tomato and lycopene on hepatic nuclear receptor-, stress-, and metabolism-related gene expression in mice.
Tan HL; Moran NE; Cichon MJ; Riedl KM; Schwartz SJ; Erdman JW Jr; Pearl DK; Thomas-Ahner JM; Clinton SK;
J Nutr; 2014; 144 (4):431-9 2014 Feb 19 PMID:24553694

FAQ

What is the best approach for determining where to set the CT threshold when you have >15 samples? Is it best to go through all of them, looking for a range of best fit, and then just choose one value that fits all of them?
The best way to set the threshold is to make sure that your PPC values are between 18 and 22. I would look my first PCR Array, set it so that the PPC is at 20, and see if the same threshold fits for the rest of the arrays.
FAQ ID -2705
How many housekeeping genes are included in each PCR Array?
Each PCR Array has 5 housekeeping genes. You can use one or an average of the most stable ones to do data analysis.
FAQ ID -2704
Are primers available that only detect mitochondrial DNA encoded genes and not nuclear genomic DNA encoded genes?
There are less than a dozen genes encoded by the mitochondrial genome (all other mitochondrial proteins are encoded by nuclear genes), and they are all transcribed as one transcript (just like any prokaryote), so distinguishing the expression of individual genes by real-time RT-PCR is not possible.
FAQ ID -2680
What is the RT² Profiler PCR Array?
The RT² Profiler PCR Array is a 96-/384-well plate or 100-well disc that contains gene-specific Primer Assays for a thoroughly researched set of relevant, pathway- or disease-focused genes. It simultaneously profiles the expression of 84 pathway-specific genes, and five housekeeping genes. Each RT² Profiler PCR Array also includes a Genomic DNA Control (GDC) assay, triplicate Reverse Transcription Controls (RTC), and triplicate Positive PCR Controls (PPC).
FAQ ID -2718
What are the guidelines for choosing a housekeeping gene for normalizing qPCR results?

If you are unsure of the correct housekeeping gene(s), review the literature and technical information in your field to determine which gene(s) other researchers commonly use. It is recommended that multiple housekeeping genes be utilized for each gene expression experiment, to account for any impact that an experimental condition may have on the expression of an individual housekeeping gene. For a systematic assessment of which housekeeping genes are appropriate for your specific experimental conditions, we recommend using the Housekeeping Genes RT2 Profiler PCR Arrays for human (330231 PAHS-000), mouse (330231 PAMM-000), or rat (330231 PARN-000). These arrays consist of 8 sets of 12 common housekeeping genes. They are a valuable tool for easily identifying genes with a constant level of expression among your different experimental conditions.

FAQ ID -2674
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
Do you always run samples in triplicates?
No. Data Analysis can be done with a little as 2 PCR Arrays. Whether or not you run a sample in triplicate is determined by experimental setup and what you are going to use the data for.
FAQ ID -2703
Is it good to pool multiple RNA replicates to detect expression changes that are consistently reproducible?
With the additional RT2 PreAMP methodology, only 1 ng of RNA is now needed for PCR Array analysis. Pooling RNA from different sources should only be done when there is not enough sample. We recommend running biological replicates.
FAQ ID -2663
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
On which instrumentation will the RT² Profiler PCR Array work?

For real-time detection, the RT² Profiler PCR Array is currently available for most QIAGEN, ABI, BioRad, Eppendorf, Stratagene, TaKaRa, Fluidigm, Cepheid, and Roche real-time instruments. Please refer to the link below, to determine which RT² Profiler PCR Array plate format is compatible with your instrument.

http://www.sabiosciences.com/manuals/PCRArrayGuide.pdf


 
FAQ ID -2719
What negative controls are typically included in qPCR and/or qRT-PCR experiments?

The 3 most common negative controls included in a qPCR and/or qRT-PCR experiment are as follows:

1. A no template control (NTC) omits any DNA or RNA template from a reaction, and serves as a general control for extraneous nucleic acid contamination. When using SYBR Green chemistry, this also serves as an important control for primer dimer formation. Within the RT2 Profiler PCR Arrays, the GDC well also serves as a no template control, as this assay is designed to detect Genomic DNA.

2. A no reverse transcriptase control (NRT) or minus reverse transcriptase control (MRT) involves carrying out the reverse transcription step of a qRT-PCR experiment in the absence of reverse transcriptase. This control assesses the amount of DNA contamination present in an RNA preparation.

3. A no amplification control (NAC) omits the DNA polymerase from the PCR reaction. This is a control for background fluorescence that is not a function of the PCR. Such fluorescence is typically attributable to the use of a degraded, dual-labeled probe. This control is unnecessary when utilizing SYBR-Green probe chemistries.

FAQ ID -2672
Do I need to run a standard curve before the actual PCR array experiment?
There is no need to run a standard curve before doing the RT2 PCR Array experiment. Usually we recommend starting with 1000 ng total RNA for a 96-well PCR array.
FAQ ID -2664
What are the most reliable methods for preparing high-quality RNA from cell or tissue samples, for use in gene expression analysis experiments?
We recommend the use of RNeasy Mini Kits. Cultured cells, and freshly isolated white blood cells, may be harvested by centrifugation, and used directly with this kit. For the isolation of high-quality RNA from animal tissues, we recommend RNeasy Plus Universal Kit.
FAQ ID -2657
What are the main differences between the qBiomarker PCR Arrays and the RT2 Profiler PCR Arrays?
The qBiomarker PCR Arrays contain gene lists that have been biologically validated and selected to measure the expression of a limited number of genes that are highly predictive for a biological process. Each qBiomarker PCR Array is designed to analyze multiple samples on the same 96-well or 384-well PCR plate. These arrays are best suited for screening and validation applications for a specific biological process. In contrast, the RT2 Profiler PCR Arrays typically have 84 pathway focused genes which are selected based on a different bioinformatic process and are best suited for gene expression profiling applications where a relative fold change result, and not a predictive answer, is necessary.
FAQ ID -2438
How can I predict the percent qPCR signal due to contaminating DNA, for a given qPCR assay, and its matching NRT control?

Assuming 100% amplification efficiency, each step increase in Ct value represents a doubling in the amount of qPCR template. Therefore, evaluating the difference in Ct values between the qPCR assay, and its matching NRT control, leads to the following predictions:

CtNRT - Ct+RT Fraction of gene expression signal due to contaminating DNA Percentage of gene expression signal due to contaminating DNA
1 (1/21) = 1/2 50%
2 (1/22) = 1/4 25%
3 (1/23) = 1/8 13%
4 (1/24) = 1/16 6%
5 (1/25) = 1/32 3%

FAQ ID -2688
How can I determine whether amplification occurs from mRNA-derived cDNA or from genomic DNA contamination?
The most rigorous method to detect genomic DNA contamination, particularly with the RT² qPCR Primer Assays, is to perform a No Reverse Transcriptase (NRT) control. The PCR will have no cDNA template derived from mRNA, and any detectable product could only have been derived from genomic DNA contamination.
FAQ ID -2687
Why are my qPCR Ct values too low (< 12) in my qRT-PCR Assay?
You may be using too much template. Use less input total RNA for reverse transcription, or use template at a greater dilution factor (lower concentration). Do not pipet a volume of less than 1 μl.
FAQ ID -2684
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
What positive controls are typically included in qPCR and/or qRT-PCR experiments?

It is critical to include appropriate positive controls in a qPCR experiment to determine if false negatives are being detected in the experiment. Positive controls fall into one of 2 classes.

1. Exogenous positive controls refer to the use of external DNA or RNA carrying a target of interest. If these positive controls are assayed in separate wells/tubes from the experimental sample, they serve as a control to determine whether or not the reverse transcription and/or PCR reaction conditions are optimal. Additionally, exogenous DNA or RNA positive controls may be spiked into the experimental sample(s), and assayed in parallel or in a multiplex format with, the target of interest. These control reactions assess whether the samples contain any components that inhibit reverse transcription and/or PCR.

2. Endogenous positive controls refer to the use of a native target that is present in the experimental sample(s) of interest, but is different from the target under study. These types of controls are often referred to as normalizers, and are typically used to correct for quantity and quality differences between samples.

Within the RT2 Profiler PCR Arrays, the Positive PCR Control (PPC) wells contain a plasmid with a primer assay that detects a sequence it produces. This allows for quick confirmation of the performance of the PCR steps.

The RTC wells include assays that detect the artificial RNA that is spiked in to each sample during the cDNA synthesis step. This ensures the Reverse Transcription step proceeded as needed.

FAQ ID -2673
Will the Reverse transcription control on the RT2 profiler PCR array work on any cDNA library?

The Reverse transcription control requires that the reverse transcription is done with the RT2 first strand kit. No other cDNA synthesis method can use this control. 

FAQ ID - 3534
Why is 18S ribosomal RNA (rRNA) used as a housekeeping gene to normalize sample-to-sample, systematic variation in qPCR assays?
18S ribosomal RNA is a widely used control for qRT-PCR analyses because of its invariant expression across tissues, cells, and experimental treatments. However, due to its extremely high expression in most cell types, it can sometimes be challenging to use 18S rRNA as an endogenous normalizer for several gene expression assays in the same reaction.
FAQ ID -2675
Can I manually set the threshold line?
You can manually set the threshold line. If you are using a catalogued PCR Array, the PPC values should be 20 +/- 2 Cts. Use the same threshold on all of your PCR Arrays.
FAQ ID -2702
What is the delta Rn value?
The Rn value, or normalized reporter value, is the fluorescent signal from SYBR Green normalized to (divided by) the signal of the passive reference dye for a given reaction. The delta Rn value is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter reliably calculates the magnitude of the specific signal generated from a given set of PCR conditions. For more information, please refer to your cycler's user manual.
FAQ ID -2681
How do you determine the efficiency using the PCR array?
We determine the amplification efficiency during wet bench testing of our assays using standard curve dilutions, or by single curve analysis. If you would like to calculate the efficiency of each curve using single curve analysis, then you can try Real-Time PCR Miner, LinReg or Dart PCR. Each of these can be found using a GOOGLE search.
FAQ ID -2701
May I try the data analysis tool without using your PCR array kit?
Yes, all you need to do is to organize your data into a “custom PCR Array” file. When you upload it to the website, use the custom PCR array name CUSTOM. The locations of the blank excel spreadsheet is: http://www.sabiosciences.com/pcrarraydataanalysis.php#custom
FAQ ID -2698