QuantiTect Primer Assays

Yes, the layout of the QuantiTect Primer Assay Plates can be seen when ordering online via GeneGlobe. However, the drag-and-drop function for manual re-positioning of individual assays on the plate is only available for the 96-well format.

We would recommend citing the name of the QuantiTect Primer assay and its source, QIAGEN.

We have performed numerous tests comparing the performance of QuantiFast Kits and QuantiTect Kits with QuantiTect Primer Assays. Due to the optimized ion concentrations in the PCR buffers, both perform equally well with QuantiTect Primer Assays and do not require any adjustment of primer concentration.

Yes, please visit our website section 'Using endogenous control genes in real-time RT-PCR' for general information. It provides a list of relative gene expression levels for commonly used human and mouse reference genes.

We offer a set of ready-to-order control genes for use in SYBR Green based as well as probe based real-time RT-PCR.

• QuantiTect Primer Assays are primer sets for use in SYBR Green based real-time RT-PCR analysis.
• QuantiFast Probe Assays are primer-probe sets for use in dual-labeled probe based real-time RT-PCR.

In addition, you may want to refer to the following citations on reference gene selection for quantitative real-time PCR:

• Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, DePaepe A, Speleman F [2002]: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:0034.

• Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A., 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313(4): 856-62.

• Katrien Smits,Karen Goossens, Ann Van Soom, Jan Govaere, Maarten Hoogewijs, Emilie Vanhaesebrouck,Cesare Galli, Silvia Colleoni, Jo Vandesompele, and Luc Peelman [2009]Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes. Dec 11;2:246.

When retrieving a QuantiTect Primer Assay for your gene of interest in GeneGlobe, you will find a link ('View Details') to the main assay. Right below, you may also see a link to previous versions of the assay and/or a link to assays for transcript variants if these are available.

We recommend choosing the main QuantiTect Primer Assay, since it has the latest version number and is designed to detect the main transcript and all possible transcript variants. You can choose a previous version of the assay if you have ordered it before and it works in your experiments. If you only want to detect a certain splice variant, choose an assay for a specific transcript variant.

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

Since we only use official gene names from Entrez to name our QuantiTect Primer Assays, the name of the assay for one species may differ from that of another species.

For example, interleukin 17 is known as IL-17 in human, and as LOC301289 in rat.

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.

Yes. QuantiTect Primer Assays are guaranteed for use with QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits at the specified annealing temperature of 60ºC. We have extensively tested many QuantiTect Primer Assays with success under these conditions.

The QuantiTect Primer Assay may have been renamed. Since we only use official gene names from Entrez to name our QuantiTect Primer Assays, names are subject to change in case an update occurs at NCBI's Entrez data base. 

For example, Hs_GYG was officially renamed by Entrez to Hs_GYG1, so we changed the name of the Primer Assay accordingly:

Hs_GYG_1_SG QuantiTect Primer Assay was renamed Hs_GYG1_1_SG QuantiTect Primer Assay.

Performing a search in GeneGlobe using the gene symbol for the gene of interest should retrieve the corresponding QuantiTect Primer Assay with the new name. To find the original name, click on the link to the assay and look under 'Other names' on the 'View Details' page.

QuantiTect Primer Assays are primer pairs designed and bioinformatically validated specifically for real-time RT-PCR with SYBR Green detection. To find a primer assay for your target gene of interest, please visit our GeneGlobe data base.

For best results, we strongly recommend using QuantiTect Primer Assays in combination with QIAGEN's products for SYBR Green-based Real-Time PCR and RT-PCR.

No. The miScript Universal Primer contained in the miScript SYBR Green PCR Kit is necessary for the detection of miRNA.

For detection of mRNA, a QuantiTect Primer Assay for the gene of interest should be used.

The primers of the QuantiTect Primer Assays are at a proprietary concentration that was specially optimized for sensitive and efficient amplification in any real time cycler. Always dilute these primers to a final work solution of 1x in your reaction, using either the QuantiTect SYBR Green PCR Kit or the QuantiTect SYBR Green RT-PCR Kit. Follow the instructions for use of the Primer Assays in the QuantiTect Primer Assay handbook.

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

This depends on the QuantiTect Primer Assay Plate format.

QuantiTect Primer Assay 96 Plate (20):

• 20 x 50 ul reactions or 40 x 25 ul reactions

QuantiTect Primer Assay 96 Plate (100):

• 100 x 50 ul reactions or 200 x 25 ul reactions

QuantiTect Primer Assay 384 Plate (20):

• 20 x 50 ul reactions or 40 x 25 ul reactions

The annealing temperature for QuantiTect Primer Assays should be 55^oC when used with the QuantiTect SYBR Green PCR Kit and the QuantiTect SYBR Green RT-PCR Kit.

Use of the QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits requires a combined annealing/extension step at 60^oC, as described in the QuantiTect Primer Assay Handbook.

Note that these Assays are guaranteed for use with the QuantiTect or QuantiFast chemistries only!

Sequences of the QuantiTect Primer Assays are not provided. Approximate location of primers within a specific gene can be viewed on the Product Detail pages retrieved via our GeneGlobe data base.

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.

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

Yes, QuantiTect Primer Assays work very well under fast-cycling conditions. Results achieved with QuantiFast SYBR Green Kits are comparable to those achieved with QuantiTect SYBR Green Kits.

For each QuantiTect Primer Assay, we retrieve the sequence of the target gene from curated databases and exclude SNP regions from assay design.

We then use our proprietary algorithm to design assays that amplify RNA sequences only (i.e., at least one primer overlaps a splice site), provided that information on the position of splice sites is available. The assays are designed to provide optimal performance with QuantiFast and QuantiTect SYBR Green Kits.

After assay design, we validate randomly selected QuantiTect Primer Assays using real-time RT-PCR to check their compatibility with various real-time cyclers. Both two-step and one-step RT-PCR are carried out. Successful amplification of the target is indicated by the following factors: high sensitivity, high efficiency, high specificity (i.e., a single peak in melting curve analysis), and no primer–dimers in the no-template control (NTC).

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

In most cases, earlier versions of the QuantiTect Primer Assays do work:

• earlier versions may detect both RNA and genomic DNA sequences, while the latest version may be specific for RNA sequences
• there may be only a difference of one nucleotide between the latest and the previous assay version
• earlier versions may not detect all transcript variants, while the latest version can

Previous assay versions can still be used if they work in your experiments. However, we recommend using the latest version. In very rare cases, original versions of the assay may not perform due to significant updates of transcript sequence information in Entrez or Ensembl, which our QuantiTect Primer Assays are based on.

(To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base).

No. All assays on a single QuantiTect Primer Assay Plate are for the same number of reactions.

QuantiTect Primer Assays for your target genes of interest can be conveniently ordered via GeneGlobe.

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.

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line.  Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

If the issue persists, please send the original run file to QIAGEN Technical Services.

QuantiTect Primer Assays are optimized for an annealing temperature of 55ºC when using the QuantiTect SYBR Green PCR Kit (for two-step RT-PCR) or the QuantiTect SYBR Green RT-PCR Kit (for one-step RT-PCR) .

Increasing the annealing temperature will reduce PCR performance (sensitivity and PCR efficiency) when using QuantiTect Primer Assays with these kits.

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher^®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

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.

For American and Canadian customers, it takes about 3 days for delivery of the QuantiTect Primer Assays.

For the rest of the world, there are 7 working days between ordering online and receiving the assay.

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

Although there may have been sequence updates in Entrez and/or Ensembl which serve as the basis for our QuantiTect Primer Assay design, the assay still matches the amplified region. Therefore, there is no need to design a new assay.

The version number of an assay appears in the assay name as in the following example:

Hs_LTA_2_SG QuantiTect Primer Assay (200)

Hs:       human

LTA:     lymphotoxin alpha (TNF superfamily, member 1)

2:         assay version 2

SG:      assay is for SYBR Green based real-time RT-PCR

(200):   200 x 50 µl reactions

Many genes have more than one transcript due to alternative splicing. We refer to the various transcripts (splice variants) for a particular gene as transcript variants. When searching GeneGlobe for a QuantiTect Primer Assay, you may locate a link below the assay name saying 'Show Primer Assays for transcript variants', whenever there are assays for transcript variants available. If there is no such link below an assay name, then the specific Primer Assay can detect all transript variants simultaneously.

Example:

Human adrenergic, alpha-1A-, receptor ADRA1A

For this specific QuantiTect Primer Assay there are assays for transcript variants available which can be accessed via the link below the QuantiTect Primer Assay name.

Assays for transcript variants are named as follows:

Hs_ADRA1A_vb.1_SG QuantiTect Primer Assay (200)

Hs:            human

ADRA1A:   adrenergic, alpha-1A-, receptor

vb.1:         transcript variant b for a specific RefSeq ID Number; assay version 1

SG:           assay is for SYBR Green based real-time RT-PCR

(200):        200 x 50 µl reactions

In this specific example, the letters 'vb.1' in 'Hs_ADRA1A_vb.1_SG' are used to indicate that the assay is specific for the transcript variant with RefSeq ID NM_033302, and that the Primer Assay is the first version for this particular transcript variant.

Click the 'View details' link for a particular assay to find out which transcript variant it is detecting.

The minimum order is 24 assays per QuantiTect Primer Assay 96 Plate, and 96 assays per QuantiTect Primer Assay 384 Plate.

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

• Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
• DNA template concentrations should be normalized using the same dilution buffer. Ensure the C_T values are below 30 and differ no more than 3 C_T values across individual samples.
• Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
• Always start with 0.7 µM primer concentration

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Reaction components for SYBR Green real-time RT-PCR must be purchased separately.

• For two-step RT-PCR, the QuantiTect Reverse Transcription Kit and QuantiTect SYBR Green PCR Kit are recommended
• For one-step RT-PCR, the QuantiTect SYBR Green RT-PCR Kit is recommended

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

For quantification of RNA, we strongly recommend using RNA molecules as standards. Use of in vitro transcripts as standards takes into account the variable efficiency of the RT reaction. An alternative to the use of in vitro transcripts as RNA standards is the use of a defined RNA preparation (e.g., from a cell line or virus preparation), for which the absolute concentration of the target has already been determined.

For quantification of DNA, several types of DNA can be used, such as plasmids, PCR products, or genomic DNA.

For more information, see Appendix E 'Generating Standard Curves' in the QuantiTect Probe PCR Handbook.

We have performed numerous tests comparing the performance of Rotor-Gene SYBR Green PCR Kits and QuantiTect Kits with QuantiTect Primer Assays. Due to the optimized ion concentrations in the PCR buffers, both perform equally well with QuantiTect Primer Assays and do not require any adjustment of primer concentration.

Yes, it is possible to order QuantiTect Primer Assay Plates by e-mailing or faxing the completed order documentation, containing order form and separate plate layout information. Note that the respective Excel file containing the plate layout information has to be sent electronically. Only the order form itself may be sent by fax.

QuantiTect Primer Assays in plate format are available for Human, Mouse, Rat, Drosophila, Arabidopsis, Chicken, and Dog, with more species to come.

QuantiTect Primer Assays can be searched for and ordered in either tube or plate format online via GeneGlobe.

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (T_m) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer T_m, but approximately 3ºC lower than the specific amplicon T_m.

We strictly recommend to use a ramp time of 2°C/sec when running QuantiTect Primer Assays on the LightCycler because cycling is much more robust when compared to using 20°C/sec for ramping. If optimal assay conditions have been established using our recommended ramp time, customers can of course try to use 20°C/sec ramping for denaturation and annealing steps at their own risk.

QIAGEN cannot guarantee that QuantiTect Primer Assays designed for mouse or human will also work for rat.

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.