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Cat. No. / ID: 60404
Cat. No. / ID: 61504
Cat. No. / ID: 9001580
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Cat. No. / ID: 9024203
Cat. No. / ID: 9025620
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.
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.
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.
The PIK3CA RGQ PCR Kit enables qualitative detection of 11 mutations in the PIK3CA gene
Based on our extensive and successful testing of many QuantiTect Primer Assays with Rotor-Gene SYBR Green PCR Kits, we guarantee this.
Reaction volumes suitable for use on the Rotor-Gene Q are:
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.
Yes, please follow the Supplementary Protocols at the links below:
'Rotor-Gene software setup for the Rotor-Gene SYBR Green PCR Kit' (PCR106)
'Rotor-Gene software setup for the Rotor-Gene SYBR Green RT-PCR Kit' (PCR107)
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.
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.
The composition of Buffer ATE is:
- 10 mM Tris-Cl pH 8.3
- 0.1 mM EDTA
- 0.04% NaN3 (sodium-azide)
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Please send the original OTV run file to QIAGEN Technical Service for further assistance.
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.
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.
The buffer composition, which affects the initial reactivation of HotStarTaq Plus DNA Polymerase, has been optimized for each respective Rotor-Gene Kit.
Yes, please choose from the Supplementary Protocols below:
QuantiFast Probe RT-PCR +ROX Vial Kit:
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.
Yes, use the assays at a final concentration of 1x with Rotor-Gene Probe Kits on the Rotor-Gene Q cycler.
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.
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
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.
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.
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.
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.
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
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.).
Yes, simply use the assays at a final concentration of 1x with the Rotor-Gene Multiplex PCR Kit.
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.
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.
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.
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.
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.
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.
The sample rotor of the Rotor-Gene Q spins continuously at a speed of 400 rpm during a run.
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.
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.