RT² RNA QC PCR Arrays

用于在PCR芯片分析前对RNA样本进行质量评估

Products

RT² RNA QC PCR Arrays 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。

RT² RNA QC PCR Array

Cat. No. / ID: 330291

RT² Profiler QC Array

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Features

检测RNA完整性、抑制剂和DNA污染
十分适用于高通量RNA质量控制
节约时间，避免浪费试剂

Product Details

RT² RNA QC PCR Arrays用于在使用标准或定制的RT² Profiler PCR Array分析前，对人类、小鼠、大鼠、狗或猕猴的RNA样本进行质量评估。RT² RNA QC PCR Arrays包含多个PCR对照，可检测RNA的完整性、逆转录和PCR扩增的抑制剂、基因组DNA和常规DNA污染。

Principle

RT² RNA QC PCR Arrays（用于人类、小鼠、大鼠、狗或恒河猴）可在使用标准或定制的RT² Profiler PCR Array分析前，对RNA样本进行质量评估。芯片包含多个PCR对照，可检测RNA的完整性、逆转录和PCR扩增抑制剂、基因组和常规DNA污染（见下面）。任何质量控制的失败都表明RNA可能导致在基于SYBR^® Green的real-time PCR中出现假阴性或假阳性结果。

RT²RNA QC PCR Arrays增加了real-time PCR结果的可信度。通过在同一孔板上同时对至多12个样本进行RNA质量控制，用于确保众多样本在整个研究中的一致性。使用RT² RNA QC PCR Array验证RNA样本质量，可避免因使用不合标准的样本浪费预混液和PCR芯片。

对照

RT² RNA QC PCR Array包括以下对照：

RNA的完整性：2个管家基因（HK1和HK2）
基因组DNA污染：基因组DNA对照（GDC）
基因组DNA污染：非逆转录对照（NRT）
非特异DNA污染：非模板对照（NTC）
逆转录抑制剂：逆转录对照（RTC）
PCR扩增抑制剂：阳性PCR对照（PPC）

需使用RT² First Strand Kit从总RNA中合成cDNA模板，以准确说明RT² RNA QC PCR Array上的所有控制元素。

规格

人类、小鼠、大鼠、狗和恒河猴的RT²RNA QC PCR Arrays提供以下规格:

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

每个cDNA模板（使用RT² First Strand Kit合成）与PCR预混液混合后，等体积加入到HK1、HK2、GDC、RTC以及PPC两孔中的一个孔中。每个RNA样本与PCR预混液混合后，加入到样本的NRT孔中。稀释的PCR预混液加入到NTC及第二个PPC孔中，然后运行real-time PCR循环程序。

Applications

人类、小鼠、大鼠、狗或猕猴的RT² RNA QC PCR Arrays非常适合在使用RT² Profiler PCR Array进行real-time PCR基因表达分析前，对RNA样本进行质量评估。

Resources

Download Safety Data Sheets for QIAGEN product components.

Data analysis file for RT² Profiler PCR Array Housekeeping Genes
Catalog number- 330231
Pathway number- PAXX-000

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

FAQ

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

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

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

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

RT2 RNA QC PCR Arrays