PIK3CA RGQ PCR Kit

For qualitative detection of 11 mutations in the PIK3CA gene by real-time PCR

Products

For Research Use Only. Not for use in diagnostics procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.

PIK3CA RGQ PCR Kit

Cat. No. / ID: 873101

For 24 reactions: 6 Reaction Mixes, Positive Control, Taq DNA Polymerase, Water for NTC, and Water for Sample Dilution

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Rotor-Gene Q 5plex HRM Platform

Cat. No. / ID: 9001580

Real-time PCR cycler and High Resolution Melt analyzer with 5 channels (green, yellow, orange, red, crimson) plus HRM channel, laptop computer, software, accessories: includes 1-year warranty on parts and labor, installation and training not included

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Rotor-Gene Q 5plex HRM System

Cat. No. / ID: 9001650

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QIAamp DNA FFPE Tissue Kit (50)

Cat. No. / ID: 56404

For 50 DNA preps: 50 QIAamp MinElute Columns, Proteinase K, Buffers, Collection Tubes (2 ml) Use our next-generation QIAamp DNA FFPE Advanced Kits for improved performance.

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QIAamp Circulating Nucleic Acid Kit (50)

Cat. No. / ID: 55114

For 50 preps: QIAamp Mini Columns, Tube Extenders (20 ml), QIAGEN Proteinase K, Carrier RNA, Buffers, VacConnectors, and Collection Tubes (1.5 ml and 2 ml)

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Features

Reliable detection of relevant mutations in the PIK3CA gene
High sensitivity and specificity
Results in less than two working days
Optimized reagents and reaction mixes
Automated data analysis using Rotor-Gene AssayManager v2.1 software

Product Details

The PIK3CA RGQ PCR Kit is a real-time qualitative PCR test for the detection of 11 mutations in the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene using a sample of DNA extracted from either formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue or K₂EDTA anticoagulated blood plasma, taken from a patient with breast cancer.

Principle

The PIK3CA RGQ PCR Kit is comprised of six reaction mixes; one control reaction targeting exon 15 and five mutation-specific reactions utilized to detect 11 mutations in exons 7, 9 and 20 of the PIK3CA gene (Exon 7: C420R; Exon 9: E542K; E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y). Allele-specific technology allows accurate and highly reproducible detection of mutations; DNA is selectively amplified using ARMS primers, probes and PCR clamps, with sensitive signal detection using the Rotor-Gene Q 5plex HRM instrument. Data analysis is performed automatically. Outputs for each PCR are displayed individually, with "Amplification Detected" displayed when a specific PIK3CA mutation is detected.

Procedure

The simple and straightforward testing workflow begins with manual DNA extraction from either FFPE breast tumor tissue (using the QIAamp DSP DNA FFPE Tissue Kit or QIAamp DNA FFPE Tissue Kit) or from K₂EDTA anticoagulated plasma (using the QIAamp DSP Circulating Nucleic Acid Kit or QIAamp Circulating Nucleic Acid Kit), followed by sensitive real-time PCR on the Rotor-Gene Q 5plex HRM instrument. Rotor-Gene AssayManager software rapidly and accurately reports results, informing the system operator if one or more of the 11 mutations detected by the kit are present in each sample. The kit is a qualitative assay that can yield results in less than two working days.

Applications

The PIK3CA RGQ PCR Kit enables qualitative detection of 11 mutations in the PIK3CA gene

Resources

Sample to Insight solutions for successful molecular analysis

High-quality, nucleic acid purification for successful PCR and NGS experiments.

Second edition — innovative tools

Now with even more applications!

Critical factors for molecular analysis of FFPE samples

For concentration and purification of free-circulating DNA and RNA from human plasma or serum

For research use only

For verification of thermal accuracy of Rotor-Gene real-time cyclers

Q546R false mutation positive result

Fast and Integrated Screening Workflow to Assess Gene Expression of Individual Pathways

For use with the Q-Rex Software to classify the genotypes of unknown samples.

Gene Expression Analysis Plug-in for the Q-Rex Software

For use with the Q-Rex Software v1.0 or higher to detect targets

For use with the Q-Rex Software to calculate absolute concentration of targets by PCR

Guideline for ISO 20836:2020

For use with Rotor-Gene Q Software version 2.3.4

For use with the Rotor-Gene Q and QIAquant instruments.

Note: The Q-Rex Software is compatible only with QIAquant instruments with serial numbers that start with 3108 (produced after 11/2021).

For use on the Rotor-Gene Q. Rotor-Gene Q software 2.3.5 is compatible with Windows 7 and Windows 10 operating systems.

Digital PCR (dPCR) is a powerful technique that detects and quantifies ultra-rare mutations in a high background of wild-type cfDNA down to 0.1% variant allele frequency. Here, we describe end-to-end manual and automated workflows that enable accurate detection and absolute quantification of ultra-rare PIK3CA variants in cfDNA using the QIAcuity Digital PCR System.

September 2023

FAQ

Based on our extensive and successful testing of many QuantiTect Primer Assays with Rotor-Gene SYBR Green PCR Kits, we guarantee this.

FAQ ID -2124

Reaction volumes suitable for use on the Rotor-Gene Q are:

Rotor-Disc 100: 30 µl x 100-wells, 10-25 µl reaction volume
Rotor-Disc 72: 0.1 ml x 72-wells, 15-25 µl reaction volume
Strip Tubes 0.1 ml: 0.1 ml x 72-wells, 10-30 µl reaction volume, strips of 4 tubes and caps
PCR Tubes 0.2 ml: 0.2 ml x 36-wells, 15-50 µl reaction volume, individual tubes with caps

FAQ ID -9030

The Rotor-Gene Q software allows creation of new excitation/detection wavelength combinations, which means that Rotor-Gene Q will work with dyes you may want to use in the future.

FAQ ID -2087

Yes, please follow the Supplementary Protocols at the links below:

Rotor-Gene SYBR Green PCR Kit:

'Rotor-Gene software setup for the Rotor-Gene SYBR Green PCR Kit' (PCR106)

Rotor-Gene SYBR Green RT-PCR Kit:

'Rotor-Gene software setup for the Rotor-Gene SYBR Green RT-PCR Kit' (PCR107)

FAQ ID -2104

RNA purified with the QIAamp Viral RNA Mini Kit has been used for quantification by qPCR with QuantiTect Probe RT-PCR Kit on QIAGEN Rotor-Gene Q.

3828

The MinElute spin columns included in the following kits should be stored at 2–8°C upon arrival: AllPrep DNA/RNA Micro, EpiTect Fast DNA Bisulfite, EpiTect Fast FFPE Bisulfite, EpiTect Fast LyseAll Bisulfite, EpiTect Plus DNA Bisulfite, EpiTect Plus FFPE Bisulfite, EpiTect Plus LyseAll Bisulfite, exoRNeasy Serum/plasma Maxi, exoRNeasy Serum/Plasma Midi, GeneRead DNA FFPE, GeneRead rRNA Depletion, GeneRead Size Selection, MinElute Gel Extraction, MinElute PCR Purification, MinElute Reaction Cleanup, miRNeasy FFPE, miRNeasy Micro, miRNeasy Serum/Plasma, QIAamp DNA FFPE, QIAamp DNA Investigator, QIAamp DNA Micro, QIAamp MinElute Media, QIAamp MinElute Virus Spin, QIAamp MinElute Virus Vacuum, RNeasy FFPE, RNeasy Micro, RNeasy Plus Micro.

Short-term storage (up to 4 weeks) at room temperature (15–25°C) does not affect the performance. However, for optimal performance and quality, storage temperature should not exceed 25°C.

FAQ ID - 3560

In the REST 2009 software, the hypothesis test performs 10,000 random reallocations of samples and controls between the groups, and counts the number of times the relative expression on the randomly assigned group is greater than the sample data.

FAQ ID -2459

Rotor-Gene Probe Kits are specially developed for Rotor-Gene cyclers. The unique rotary system of the cyclers combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Probe Kits do not contain ROX dye.

FAQ ID -2125

Both types of tubes run on the Rotor-Gene Q, and can be labeled on the top without any interference with data acquisition, because signal is detected from the bottom of the tube. Labeling is helpful and can prevent possible sample mix up.

FAQ ID -2082

There are several features available to allow exporting data across platforms to a variety of software packages. Sample names can easily be imported into the Rotor-Gene Q Excel sample sheets. By using a function "Export to Excel" the software automatically exports any results table in the software to Excel. Furthermore, reports can now be exported as Word files, which make the data compatible with any Microsoft office/Mac application. Reports can also be saved or sent in HTML format. All figures and graphs can be exported as Jpeg or Bitmap files for use in any desktop publishing software.

FAQ ID -2089

No. The master mixes in Rotor-Gene Kits contain dTTP instead of dUTP. If UNG treatment is required, we recommend using QuantiTect +UNG Kits. QuantiTect Kits are also compatible with the Rotor-Gene Q; however, the kits require a significantly longer cycling time.

FAQ ID -2117

In the REST 2009 software, the range for the Standard Error on the results tab is 68% C.I. (confidence interval).

FAQ ID -2455

No. The fixed optical path ensures uniform illumination and detection from sample to sample eliminating the need to use a reference dye such as ROX on the Rotor-Gene Q.

FAQ ID -2079

Yes. The sample sheet can be amended during or after a PCR run by activating the 'Edit samples' button in the Rotor-Gene Q software. The sample sheet will open and may be modified.

FAQ ID -2092

Yes, data can be analyzed on the Rotor-Gene Q while a run is in progress, allowing to save time for planning and setting up new experiments. Multiple copies of the Rotor-Gene Q software can be opened simultaneously during a run allowing analysis of previous experiments while the system is running.

FAQ ID -2091

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.

FAQ ID -1141

Please make data are collected in the appropriate fluorescent channel. Also check the gain is optimized.

If the issue persists, please send the original run file with extension .rex to QIAGEN Technical Service for further assistance.

FAQ ID -9023

The reaction volume to be entered in the Rotor-Gene Q software should be the sum of the PCR reaction volume and the volume of Vapor-Lock.

FAQ ID -2150

At the selected data acquisition step of the thermal cycle on the Rotor-Gene Q, the fluorescent signal intensity is measured 20 times for each sample as they spin past the detector. Tubes on a rotor spin past the excitation/detection optics every 150 milliseconds. The average of the 20 readings is then taken as the fluorescence of the sample at that cycle number.

FAQ ID -2083

Please send the original OTV run file to QIAGEN Technical Service for further assistance.

FAQ ID -9022

This type of cycling allows a significant reduction in cycling time for Rotor-Gene SYBR Green PCR Kits. It is more effective than reducing the individual times for annealing and extension.

FAQ ID -2122

We strongly recommend use of the dedicated Rotor-Gene Q Accessories, e.g., PCR Tubes 0.2 ml or Strip Tubes and Caps 0.1 ml provided by QIAGEN. These tubes are designed to give low background and perfectly match the Rotor-Gene Q requirements.

FAQ ID -2085

The buffer composition, which affects the initial reactivation of HotStarTaq Plus DNA Polymerase, has been optimized for each respective Rotor-Gene Kit.

FAQ ID -2118

Yes, please choose from the Supplementary Protocols below:

QuantiFast Probe RT-PCR Kit:

QuantiFast Probe RT-PCR +ROX Vial Kit:

FAQ ID -2105

An Investigator Quantiplex Pro or Quantiplex Pro RGQ run of 40 cycles takes ca. 60 minutes.
For Investigator Quantiplex on the Rotor-Gene Q, a run of 40 cycles takes ca. 55 minutes. On the Applied Biosystems 7500 Real-Time PCR System, a run of 40 cycles takes ca. 57 minutes.

FAQ ID -2566

The Rotor-Gene Q instrument can be used with two different rotor formats: using tubes or rotor-dics. Tubes can be run in 36 and 72-well rotors and rotor-discs in Rotor-Disc 72 and Rotor-Disc 100 rotors. When programming the temperature profile please make sure the correct rotor type is selected.

FAQ ID -1519

Yes, use the assays at a final concentration of 1x with Rotor-Gene Probe Kits on the Rotor-Gene Q cycler.

See trademarks.

FAQ ID -2126

Our unique multiplex PCR buffer system with ammonium and potassium ions and Factor MP has been further optimized in QuantiFast and Rotor-Gene Kits. We have also discovered Q-Bond, a buffer component which supports the rapid formation of the polymerase–primer–template complex, leading to reduced annealing times.

FAQ ID -1430

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

The Investigator Quantiplex Kit has been validated for the Rotor-Gene Q, and the Applied Biosystems 7500 Fast and 7500 Real-Time PCR Systems.

FAQ ID -2567

Based on the defined standards present, the automatic threshold function of the Rotor-Gene Q scans through all the possible threshold levels until the best fit is determined. This is defined as the R value or correlation coefficient, which is maximized to most closely approach 1.0. If there is no standard present, the threshold can also be set manually.

FAQ ID -2088

Using the Investigator Quantiplex on the Rotor-Gene Q: All versions of the Rotor-Gene Q software can be used to run the Investigator Quantiplex.

FAQ ID -2568

No, that is not necessary. Simply use the primer concentrations specified in the protocols in the handbook supplied with your Rotor-Gene Multiplex PCR Kit.

FAQ ID -2128

For hardware related issues, please send the support package to QIAGEN Technical Service. Within the Rotor-gene Q software, click Help and select Send Support Email. In the new window, select the file that relates to the issue and email it to QIAGEN Technical Service.

FAQ ID - 9024

There are several manufacturers of high-quality real-time cyclers. These include QIAGEN's Rotor-Gene Q, Applied Biosystems, BioRad, Stratagene, Eppendorf, Roche, TaKaRa, Fluidigm, and Cepheid. The important thing to keep in mind is that, once you select an instrument to use, you must use compatible Rotor-Gene Discs and tubes, 96 or 384 well plates, and qPCR master mixes that are optimized for use in that particular instrument. For example, QIAGEN's Rotor-Gene SYBR Green, Probe, and Multiplex real-time master mixes.

FAQ ID -2670

QIAGEN recommends the annual inspection service on Rotor-gene instruments, during which all application-critical modules of the Rotor-gene are inspected and tested and an OTV check is conducted. Performed tests and test results are documented in a GMP/GLP-compliant Report. In addition, the end users can perform the temperature calibration in the lab as needed using the Rotor-Disc OTV kit.

Note: The Rotor-Disc OTV kit requires the Rotor-Disc 72 Rotor and Rotor-Disc 72 locking ring.

FAQ ID -9025

Yes. If the reaction efficiency between two PCR runs is not expected to vary, importing a standard curve from a previous run allows to estimate concentrations when a standard curve for the current run is not available. Curves can be imported from another channel, or from another run by clicking on 'Import Curve' in the Rotor-Gene Q software. Standard curves can be adjusted such that only the efficiency of the source curve is imported into the current run. Whether a standard curve should be adjusted depends on the PCR chemistry used. To adjust a standard curve, use a reference with a known concentration in the target run.

FAQ ID -2093

The majority of the Rotor-Gene Kit data shown in our literature has been generated with the help of the QIAgility instrument. We did not observe any problems during the pipetting steps.

FAQ ID -2116

The Rotor-Gene Q software version v2.2 and lower versions require a computer with Windows XP or Vista operating systems. The Rotor-Gene Q software v2.3 or higher versions can run with Windows XP or Windows 7 32-bit and 64-bit operating systems.

Rotor-Gene 6000 software requires a computer with Windows XP or Vista operating system. QIAGEN will no longer support Rotor-Gene 3000 instruments and Rotor-Gene 6.0 software starting from January 1, 2014

FAQ ID -9021

Small amounts of RNA and DNA may be difficult to measure spectrophotometrically. Fluorometric measurements, or quantitative RT-PCR and PCR are more sensitive and accurate methods to quantify low amounts of RNA or DNA.

Fluorometric measurements are carried out using nucleic acid binding dyes, such as RiboGreen® RNA Quantitation Reagent for RNA, and PicoGreen® DNA Quantitation Reagent for DNA (Molecular Probes, Inc.).

FAQ ID -728

Yes, simply use the assays at a final concentration of 1x with the Rotor-Gene Multiplex PCR Kit.

See trademarks.

FAQ ID -2131

Vapor-Lock is fully compatible with the QIAgility instrument for high-precision, automated reaction setup. It is also highly suited for use with the Rotor-Gene Q cycler. For support to program your QIAgility instrument for use with Vapor-Lock, please contact your local QIAGEN Technical Support Department.

FAQ ID -2153

Use of endogenous control genes corrects for variation in RNA content, variation in reverse-transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA sample, variation in nucleic acid recover, and differences in sample handling. The endogenous control gene ought to have consistent expression levels between samples and among treatment conditions, and ideally has a similar expression level to that of the genes of interest. Genes commonly used as references can be found at the QuantiTect Primer Assays as endogenous controls.

FAQ ID -9027

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.

FAQ ID -2123

On the Rotor-Gene Q, setting the threshold in the Yellow channel to an absolute value of 0.05 will give satisfactory results in most cases.

FAQ ID -2607

According to an Interview with Professor Dennis Lo published in QIAGEN News Molecular Diagnostics, Issue No. 5, 2002, healthy individuals have about 500-1000 genome equivalents (DNA) per ml serum/plasma.

For free-circulating DNA in plasma, the concentration can range from 1–100 ng/ml in healthy individuals.

FAQ ID -635

Rotor-Gene Kits are specifically developed for the Rotor-Gene Q PCR Cycler. The unique rotary system of the cycler combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Kits do not contain ROX dye since no normalization to a passive reference is required. Also, Rotor-Gene Kits do not contain dUTP; therefore, UNG pretreatment is not possible.

FAQ ID -2119

No. The speed at which the samples spin on the Rotor-Gene Q is not high enough to apply any significant centrifugal force on them.

FAQ ID -2081

The sample rotor of the Rotor-Gene Q spins continuously at a speed of 400 rpm during a run.

FAQ ID -2080

The specific features of Rotor-Gene Kits and Rotor-Gene cyclers work synergetically to enable an ultrafast-cycling protocol. We do not guarantee that the performance of the Rotor-Gene Multiplex RT-PCR Kit with the same cycling protocol will be the same on other cyclers.

FAQ ID -2142

Yes. Rotor-Gene Kits will also work on the Rotor-Gene 6000 and Rotor-Gene 3000 PCR cyclers with the cycling conditions specified in the Rotor-Gene kit handbooks.

FAQ ID -2121

Yes, all empty positions in the rotor of the Rotor-Gene Q have to be filled with empty tubes. This guarantees optimal temperature repartition in the Rotor-Gene Q chamber.

FAQ ID -9029