RT2 Profiler PCR Arrays
For pathway-focused gene expression analysis using laboratory-verified assays
For pathway-focused gene expression analysis using laboratory-verified assays
With the sensitivity of the RT2 First Strand Kit, as little as 1 ng or as much as 5 µg of total RNA per array plate provides greater than 80% present call rates (see figure " Positive results with as little as 25 ng RNA").
The complete PCR array system demonstrates strong correlations across technical replicates, lots, and instruments with average correlation coefficients >0.99, ensuring reliable detection of differences in expression between biological samples (see figure " High reproducibility among different users").
The PCR array system, with high-quality input RNA, yields single bands of the predicted size without primer-dimers or other secondary products, therefore providing the highly accurate real-time PCR results (see figure " A single gene-specific product in every reaction").
Uniform PCR amplification efficiency is required for the PCR array technology to allow accurate comparisons of gene expression across all genes and all samples. The unique combination of our proprietary primer design algorithm and rigorous testing of every primer assay guarantees the high performance of every primer assay on PCR arrays (see figure " PCR arrays yield highly accurate results").
RT² Profiler PCR Arrays are tested and optimized in combination with the RT² SYBR Green qPCR Mastermixes and the RT² First Strand Kit. This testing means that RT² Profiler PCR Array performance is guaranteed when all three of these components are used together.
RT2 Profiler PCR Arrays are reliable tools for analyzing the expression of a focused panel of genes. Each 96-well plate, 384-well plate or 100-well disc PCR array includes SYBR® Green-optimized primer assays for a thoroughly researched panel of relevant, pathway- or disease-focused genes. RT2 Profiler PCR Arrays can also be customized to contain a panel of genes tailored to your specific research interests. The high-quality primer design and RT2 SYBR® Green qPCR Mastermix formulation enable the PCR array to amplify 96 or 384 different gene-specific products simultaneously under uniform cycling conditions.
This combination provides the RT2 Profiler PCR Array with the specificity and the high amplification efficiencies required for accurate real-time SYBR® Green results. PCR arrays are easy to use in any research laboratory.
RT2 Profiler PCR Arrays are sensitive enough for use with RNA prepared from regular samples (0.1–5 µg RNA), FFPE samples, and small samples (1–100 ng RNA). >
Simply mix the cDNA template with the appropriate ready-to-use PCR mastermix, aliquot equal volumes to each well of the same plate, and then run the real-time PCR cycling program (see flowchart " Simple procedure"). RT2 Profiler PCR Arrays are compatible with all QIAGEN, ABI, Bio-Rad, Eppendorf, Roche, and Stratagene instruments.
RT2 Profiler PCR Arrays are available in 96-well plate, 384-well plate, and 100-well disc formats, and are used to monitor the expression of 84 or 370 genes related to a disease state or pathway, plus 5 housekeeping genes. Each RT2 Profiler PCR Array also includes control elements for:
Data can be analyzed using an easy-to-use Excel-based data analysis template or Web-based software. Data analysis is based on the ΔΔCT method with normalization of the raw data to either housekeeping genes.
RT² PCR Profiler Arrays can be used in all areas of biological and medical research, including:
Data analysis file for RT² ProfilerRT² Profiler™ PCR Array RT2 RNA QC
Catalog number- 330231
Pathway number- PAXX-999
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.
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:
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.
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.
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%|
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.
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.
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.