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RNA QC PCR Arrays

For quality assessment of RNA samples before characterization using PCR arrays

Features

  • Tests for RNA integrity, inhibitors, and DNA contamination
  • Especially useful for high-throughput RNA quality control
  • Saves time and avoids potential reagent wastage

Products

RNA QC PCR Arrays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
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RT2 RNA QC PCR Array

Cat. No. / ID: 330291

RT2 Profiler QC Array

Product Details

RNA QC PCR Arrays are designed to assess the quality of chicken, Chinese hamster, cow, dog, fruit fly, horse, human, mouse, pig, rabbit, rat, Rhesus macaque or zebrafish RNA samples before characterization with a cataloged or custom RT² Profiler PCR Array. The arrays contain a number of PCR controls that test for RNA integrity, inhibitors of reverse transcription and PCR amplification, and genomic and general DNA contamination.

Principle

RNA QC PCR Arrays (for chicken, Chinese hamster, cow, dog, fruit fly, horse, human, mouse, pig, rabbit, rat, Rhesus macaque or zebrafish) are designed to assess the quality of RNA samples before characterization with cataloged or custom RT2 Profiler PCR Arrays. The arrays contain a number of PCR controls for RNA integrity, inhibitors of reverse transcription and PCR amplification, genomic and general DNA contamination (see below). Failure of any of these controls indicates that the RNA could lead to false negative or false positive results in SYBR® Green-based real-time PCR.

RT2 RNA QC PCR Arrays offer added confidence in real-time PCR results. They are especially useful for ensuring the consistency of a large number of samples across an entire study by performing RNA quality control on up to 12 samples on one plate simultaneously. Using an RT2 RNA QC PCR Array to test the quality of RNA samples avoids potentially wasting mastermix and PCR arrays on substandard samples.

Controls

The RT² RNA QC PCR Array includes controls for:

  • RNA integrity: Two housekeeping genes (HK1, HK2)
  • Genomic DNA contamination: Genomic DNA control (GDC)
  • Genomic DNA contamination: No reverse transcription control (NRT)
  • General nonspecific DNA contamination: No-template control (NTC)
  • Reverse transcription inhibition: Reverse transcription control (RTC)
  • PCR amplification inhibition: Positive PCR control (PPC)

An RT² First Strand Kit must be used to synthesize cDNA template from total RNA in order to accurately interpret all control elements on the RT² RNA QC PCR Array.

Formats

The human, mouse, rat, dog, and Rhesus macaque RT2 RNA QC PCR Arrays are available in the following formats.

  • 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

Each cDNA template (generated using a RT² First Strand Kit) is mixed with the PCR master mix and equal volumes of each aliquotted to the HK1, HK2, GDC, RTC, and one of the two PPC wells of each sample’s column. Each RNA sample is mixed with PCR master mix and added to the samples’ NRT well. Diluted PCR master mix is added to the NTC and second PPC wells before the real-time PCR cycling program is run.

Applications

Chicken, Chinese hamster, cow, dog, fruit fly, horse, human, mouse, pig, rabbit, rat, Rhesus macaque or zebrafish RT² RNA QC PCR Arrays are highly suited to assess the quality of RNA samples before characterization using an RT2 Profiler PCR Array in real-time PCR-based gene expression analysis.

Resources

Brochures & Guides (1)
Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Kit Handbooks (1)
For quality control analysis prior to gene expression profiling using real-time RT-PCR
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