RT2 qPCR Primer Assays

For accurate and reliable gene expression analysis using laboratory-verified assays

Features

  • Complete genome coverage for multiple species
  • Amplifies a single amplicon with uniform PCR efficiency
  • Less than 5 minutes hands-on time

Products

RT2 qPCR Primer Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Image
RT2 qPCR Primer Assay (200)

Cat. No. / ID: 330001

RT2 qPCR Primer Assay

Product Details

RT² qPCR Primer Assays are specifically designed and experimentally verified for real-time PCR analysis. The rigorous assay verification criteria ensure PCR specificity and efficiency for reliable and accurate gene expression analysis results.

Principle

RT² qPCR Primer Assays use SYBR® Green-based quantitative real-time PCR technology to provide a sensitive and reliable tool for gene expression analysis.

Each assay utilizes a proprietary and experimentally verified algorithm for the design of gene-specific qPCR primers with uniform PCR efficiency and amplification conditions. Each lot of every assay is further wet-bench tested for real-time PCR performance for specificity and amplification efficiency. Amplification of a single product of the correct size with high PCR efficiency (>90%) is guaranteed when the assays are used with RT² SYBR® Green qPCR Mastermixes. The uniform PCR efficiencies and PCR conditions of the RT² qPCR Primer Assays provide an accurate and scalable solution for multiple gene expression analyses.

Applications

RT2 qPCR Primer Assays are highly suited for applications including validation of microarray-derived gene expression data, confirming gene expression knockdown by RNAi, identifying and confirming disease-associated biomarkers, and monitoring phenotypic changes related to gene expression. Multiple RT2 qPCR Primer Assays can also be used to examine a focused panel of genes.

Resources

Scientific Posters (1)
Poster for download
Kit Handbooks (1)

For gene expression analysis by real-time RT‑PCR

Instrument Technical Documents (1)
For pathway-focused gene expression analysis
Brochures & Guides (1)
Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow

FAQ

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
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
Why are my qPCR Ct values too high (> 35 or not detectable) in my qRT-PCR assay?

There are several reasons for not seeing a PCR product.

1. The corresponding gene may not be expressed above the limit of detection of the qRT-PCR assay method.

2. There may have been experimental error, in which case, use a template known to contain the gene of interest as a positive control to troubleshoot the PCR reagents and experimental procedure.

3. The RNA may have been of poor quality, in which case, be sure to perform all of the recommended quality control checks on the RNA sample (see Sample Preparation FAQs, above).

4. There may not have been enough template, in which case, use more input total RNA, or use the template at a lower dilution factor (higher concentration), or use a larger volume of template.

5. Another possible explanation pertains to when one is trying to detect cellular expression from an exogenous vector that has been introduced into a cell. If the vector expresses only the open reading frame (ORF) of the gene of interest, and the qPCR primers being used amplify a target within the 5' or 3' UTR (untranslated region) of the gene, the transcript will not be detected.

FAQ ID -2685
What is the recommended amount of input template for each RT² qPCR Primer Assay?
The useful range of input total RNA for the first strand cDNA template synthesis (reverse transcription) reaction is between 100ng and 5 µg. For initial experiments, we recommend using between 0.5 to 1 µg of input total RNA, and using 1 µl of either undiluted template or template pre-diluted 1:10 for each 25-µl RT² qPCR Assay reaction.
FAQ ID -2716
How do I determine the amplification efficiency of my qPCR assay?

Prepare five (5) 2-fold, 5-fold, or 10-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values. In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. Confirm that the correlation coefficient (R2) is 0.99 or greater. The closer the slope of this straight line is to -3.32, the closer the amplification efficiency is to 100 percent.

The amplification efficiency = [10(-1/slope)] - 1

Alternatively, a number of data analysis models have been developed that enable the calculation of PCR amplification efficiencies from individual amplification plots, without the use of standard curves. These include the Data Analysis for Real-time PCR (DART-PCR), LinReg, and the Real-time PCR Miner algorithms. Because these methods do not require the generation of standard curves, they are well suited for large scale experiments

FAQ ID -2694
What is the standard curve method for qPCR assay data analysis? How is the standard curve method for qPCR assay data analysis performed?

When using the standard curve method, the quantity of each experimental sample is first determined using a standard curve, and is then expressed relative to a calibrator sample.

In order to use this quantification method, prepare five (5) 2-fold, 5-fold, or 10-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values.

In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. Confirm that the correlation coefficient (R2) for the line is 0.99 or greater.

This plot is then used as a standard or calibration curve for extrapolating relative expression level information for the same gene of interest in unknown experimental samples. The relative quantification calibration curve result for the gene of interest is normalized to that of a housekeeping gene in the same sample, and then the normalized numbers are compared between samples to get a fold change in expression.

A standard or calibration curve must be generated separately for each gene of interest and each housekeeping gene.

 

See Critical Factors for Successful Real-Time PCR for additional details.

FAQ ID -2691
What do I need to complete a RT² qPCR Primer Assay?

You need:

  1. A RT² SYBR Green Mastermix that matches the qPCR instrument in your laboratory;
  2. RT² qPCR Primer Assays for your target genes;
  3. A Housekeeping gene RT² qPCR Primer Assay.

We also recommend using our RT² First Strand Kit for reverse transcription.

FAQ ID -2707
Why do I need to identify my real-time instrument model when placing my order for RT² qPCR Primer Assays?
The performance of our RT² qPCR Primer Assays have been tested, and are guaranteed with, our RT² SYBR Green Mastermixes only. Different master mixes have been optimized and are available for different qPCR instrumentation, because each instrument uses a different reference dye to normalize their optics. In order to guarantee that your RT² qPCR Primer Assays will work right the first time in your hands, we need to make sure that you receive the correct RT² SYBR Green Mastermix for your real-time instrument.
FAQ ID -2713
How important is the RNA purification process, for obtaining reliable qRT-PCR results?

The most important prerequisite for any gene expression analysis experiment is the preparation of consistently high-quality RNA from every experimental sample. Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment.

Genomic DNA contamination in an RNA sample compromises the quality of gene expression analysis results. The contaminating DNA inflates the OD reading of the RNA concentration. It is also a source of false positive signals in RT-PCR experiments.

RNase contamination degrades RNA samples whichcauses low signal and false-negative results in PCR.

Residual polysaccharides, collagen, other macromolecules, and organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.

For fast purification of high-quality RNA we recommend QIAGEN’s RNeasy Kits like the RNeasy Mini Kit, the RNeasy Plus Universal Kit, or the RNeasy FFPE Kit.

FAQ ID -2655
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
What is the difference between Absolute Quantification and Relative Quantification in qPCR, using the standard curve approach?

Absolute Quantification determines expression levels in absolute numbers of copies. Relative Quantification determines fold changes in expression between two samples. In absolute quantification, the precise amount of the message or template used for the curve is known. In relative quantification, the template is simply known to contain the message of interest in high abundance, but its absolute amount is not necessarily known. Unknowns are compared to either standard curve and a value is extrapolated. The absolute quantification standard curve provides the final answer. The relative quantification calibration curve result for the gene of interest is normalized to that of a housekeeping gene in the same sample, and then the normalized numbers are compared between samples to obtain a fold change.

 

See Critical Factors for Sucessful Real-Time PCR for more information.

FAQ ID -2692
Which qPCR instrument should I use with your RT² qPCR Primer Assays?

Our RT² qPCR Primer Assays may be used on any real-time instrument. qPCR solutions are available for the most popular qPCR instrumentation, including those from QIAGEN, ABI, BioRad, Stratagene.

Instrument-specific protocols are available for selected instruments, and can be accessed at the following link: http://www.sabiosciences.com/pcrarrayprotocolfiles.php

FAQ ID -2714
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
What is the comparative or ??Ct method for qPCR assay data analysis? How is the comparative or ??Ct method for qPCR assay data analysis performed?

In the comparative or ΔΔCt method of qPCR data analysis, the Ct values obtained from two different experimental RNA samples are directly normalized to a housekeeping gene and then compared. This method assumes that the amplification efficiencies of the gene of interest and the housekeeping genes are close to 100 percent (meaning a standard or calibration curve slope of -3.32) First, the difference between the Ct values (ΔCt) of the gene of interest and the housekeeping gene is calculated for each experimental sample. Then, the difference in the ΔCt values between the experimental and control samples ΔΔCt is calculated. The fold-change in expression of the gene of interest between the two samples is then equal to 2^(-ΔΔCt).

 

See Critical Factors for Successful Real-Time PCR for more information.

FAQ ID -2693
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
How do I determine the linear dynamic range of my qPCR or qRT-PCR assay?
Prepare five (5) 10-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values. In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. The linear range of this plot is the linear dynamic range of the qPCR assay.
FAQ ID -2696
What would happen if I used home-made PCR master mixes or master mixes from other manufacturers with the RT² products?
We can only guarantee the performance of RT² qPCR Primer Assays with our RT² SYBR Green Mastermixes. Our master mix components and primer design algorithm were optimized together to guarantee production of single bands of the predicted size. When we do test other sources of master mix with our Primer Assays, we frequently see primer dimers and other non-specific products that confound SYBR-Green based qPCR detection.
FAQ ID -2715
Why do I see low, poor, or sub-standard amplification efficiency in my qRT-PCR assay?
The template that you chose to use in generating your standard curve may not express your gene of interest abundantly enough to be detected after the several 10-fold serial dilutions required for the standard curve. In such a case, many of the standard curve reactions should be yielding high Ct values (> 30). You can lower the serial dilution factor to 2-fold, and generate a new standard curve. You can also try using an alternate source of template for the standard curve reactions, such as cDNA derived from a universal source of RNA, cDNA derived from a full-length in vitro transcript of the gene of interest, or even a full-length cDNA clone of the gene of interest.
FAQ ID -2695
How does QIAGEN control the quality of the RT² qPCR Primer Assays?
Each RT² qPCR Primer Assay is validated with both a real-time and conventional PCR quality control assay. These assays are carried out using a single source of genomic DNA. In order to pass QC, each primer assay must generate a single band of the correct predicted size by agarose gel electrophoresis and a single peak in the real-time dissociation curve without the appearance of primer dimers. The amplification efficiencies (DART method) and sensitivities of each primer set are also validated.
FAQ ID -2711
Why should I use RT² SYBR Green Mastermix with RT² qPCR Primer Assays?
The performance of our RT² qPCR Primer Assays have been tested, and are guaranteed with, our RT² SYBR Green Mastermixes only. Our primer design algorithm accounts for our master mix buffer system parameters such as ionic strength and magnesium chloride concentration. We have not tested our Primer Assays with other sources of master mix and their buffer system parameters. We only guarantee that our primers will perform optimally with our master mixes. We do not guarantee optimal performance with other sources of master mix, without requiring further optimization studies by the end-user.
FAQ ID -2706
Why do my qPCR amplification curves or plots decrease in fluorescence intensity after the saturation phase?
The optics of the qPCR instrumentation may be saturating due to improper instrument settings. Please consult with your instrument manufacturer for more details.
FAQ ID -2689
Why are the RT² qPCR Primer Assays not designed to cross exon-intron junctions or boundaries?
The RT² qPCR Primer Assays are not designed to cross exon-intron junctions or boundaries because for SYBR Green-based qPCR detection, the most important parameter for primer design is the generation of only a single gene-specific amplicon with high amplification efficiency, without the production of primer-dimers. Primer assays amplifying short products contained within a single exon meet this parameter most optimally. Primers that cross exon-intron junctions may still detect processed pseudogenes, heteronuclear RNA (hnRNA), as well as unannotated alternative transcripts and splice variants, thus complicating SYBR Green-based qPCR detection.
FAQ ID -2710
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 obtain the sequence of the RT² qPCR Primer Assay that I purchased?

On the product information sheet, we include the reference position for the gene-specific amplicon relative to the RT² qPCR Primer Assay's corresponding RefSeq number. Journals accept the catalog number and the reference position for publication purposes.

Customers may also use the reference position, the RefSeq number, and the NCBI database to insure that the amplicons are indeed gene-specific and even determine the approximate region amplified by the primers. For extra assurance, you can TA-clone the PCR product and then sequence it.

FAQ ID -2709
What does the RT² qPCR Primer Assay product information mean when it says that it recognizes another transcript of the same gene?
When a RT² qPCR Primer Assay recognizes another transcript of the same gene, the resulting signal by real-time or end-point detection represents the sum of the relative expression of all of the transcripts detected by the Primer Assay. When alternative transcripts or splice variants are known and annotated in the public databases, the RT² qPCR Primer Assay are designed to generate an amplicon in common to as many of those known transcripts as possible
FAQ ID -2712
What RT² qPCR Primer Assays are available?
RT² qPCR Primer Assays are available for any gene in the human, mouse, or rat genome. In addition, we also support custom primer designs for other species. You may call our Technical Support Team at 1-888-503-3187, in order to receive a quote for the design and manufacture of custom primers.
FAQ ID -2708
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
Why is my no template control (NTC) real-time Ct value < 35 cycles in my qPCR Assay?
There is DNA contamination somewhere in your PCR assay system. Use only PCR-grade reagents and lab ware. Wear gloves throughout the procedure. Always use fresh pipette tips, water and other reagents. Do not leave lab ware (open tubes and tip boxes) exposed to the air for long periods of time. The most common source of DNA contamination comes from the PCR products of previous experiments. Avoid the spread of any PCR products into the air of your working environment. Close all tubes containing PCR products once you are finished adding or removing volumes. Discard all tips or tubes that have been in contact with PCR products into a container of bleach. Clean your bench and your pipettors often. Some researchers expose lab ware with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers.
FAQ ID -2686
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70 ºC?

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

 

 

FAQ ID -2690