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PI3K-AKT Signaling Pathway Mutation PCR Array

For detecting 87 mutations in the PI3K-AKT pathway

 

Profile Mutations
 
qBiomarker Somatic Mutation PCR Array: Human PI3K-AKT Signaling Pathway
The Human PI3K-AKT Signaling Pathway qBiomarker Somatic Mutation PCR Array is a translational research tool that allows rapid and accurate profiling of the somatic mutation status for key genes in the PI3K-PTEN pathway: AKT1, AKT3, PIK3CA, PIK3R1, PIK3R5, PTEN, and STK11/LKB1. Components in this pathway are frequently mutated in human cancers (mutated in >30% solid tumor types) and therefore warrant extensive investigation to enhance the understanding of carcinogenesis and identify potential drug targets. The utility of individual and multiple somatic mutation status information in identifying key signaling transduction disruptions has been demonstrated in numerous research studies. For example, the mutation status of the EGFR and KRAS genes can predict the physiological response to certain drugs targeting these molecules. The Human PI3K-AKT Signaling Pathway qBiomarker Somatic Mutation PCR Array, with its comprehensive content coverage, is designed for studying mutations in the context of the PI3K-Akt pathway and provides the potential to discover and verify drug target biomarkers for a variety of human cancers involving the PI3K-Akt signaling pathway and downstream effectors. This array includes 87 DNA sequence mutation assays designed to detect the most frequent, functionally verified, and biologically significant mutations in the PI3K-AKT pathway. These mutations were chosen from curated, comprehensive, somatic mutation databases and peer-reviewed scientific literature. The simplicity of the product format and operating procedure allows routine somatic mutation profiling in any research laboratory with access to real-time PCR instruments.

The qBiomarker Somatic Mutation PCR Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

 

Assay Functional Annotations How It Works References Resources
 

AKT genes:
The mutation assays detects the best known AKT mutation, c.49G>A, p.E17K (AKT1; a PH domain mutation that results in constitutive targeting of AKT1 to plasma membrane), the activating mutations c.145G>A, p.E49K (AKT1) and c.511G>A, p.G171R (AKT3).

PIK3CA (phosphatidylinositol 3-kinase catalytic subunit) gene:
The mutation assays covered on this panel can detect 37 most frequently occurring PIK3CA mutations that are mainly clustered in the kinase domain (activating mutations) and in the P539-E545 region and vicinity, which are helical domain mutations that mimic activation by growth factors. The assays allow a comprehensive profiling of PIK3CA mutations that have been identified in a variety of cancers.

PIK3R1 and PIK3R5 (PI3K regulatory subunit) genes:
Two recurrent disinhibitory (c.1126G>A, p.G376R and c.1690A>G, p.N564D) mutations in PIK3R1 and a recurrent oncogenic mutation in PIK3R5 are included.

PTEN gene:
Included on the panel are 33 most commonly detected PTEN loss-of-function mutations that are mainly due to truncation or point mutation-caused phosphatase inactivation.

STK11/LKB1 gene:
Included on the panel are 11 most commonly detected STK11/LKB1 inactivation mutations that are mainly due to truncation or point mutation.

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Assay Functional Annotations How It Works References Resources
 

Overview of the qBiomarker Somatic Mutation PCR Array Layout
qBiomarker Somatic Mutation PCR Arrays are designed to test samples against a panel of mutations. The panels of mutations are selected from the same gene, biological pathway or association with a specific cancer. With different combinations of genes and mutations, each array layout can be slightly different from another. However, three types of assays are present on every array. First, bench-verified mutation specific assays that test for the presence of a single specific mutation. Second, gene copy number reference assays to normalize for DNA input for each sample. Third, Positive PCR Controls (SPC) to test for PCR inhibitors that would affect the results of the experiment.

qBiomarker Somatic Mutation PCR Array Layout (96 well format). In this example array, Wells A1 through H1 contain assays for somatic mutations in the same biological pathway, cancer type or gene. Wells H2 through H10 contain gene copy number reference assays to normalize mutation assay data. Depending on the specific array content, slight variations in plate layout can occur. For example, gene-focused arrays can often analyze two or four samples per plate. Wells H11 and H12 contain replicate Positive PCR Controls (SPC) to test for the presence of inhibitors in the sample or efficiency of the polymerase chain reaction itself using a predispensed artificial DNA sequence and the primer set that detects it. The arrays are also available as 100-well rings and 384-well plates.

qBiomarker Somatic Mutation PCR Array / Assay Protocol.
The qBiomarker Somatic Mutation Array protocol follows standard realtime PCR protocol. The experiment begins with the isolation of genomic DNA from desired samples. QIAGEN recommends that users isolate genomic DNA from samples by using the QIAamp DNA Mini Kit, QIAamp DNA FFPE Tissue Kit or DNeasy Blood & Tissue kit. Since DNA quality may have significant effects on the experimental analysis, QIAGEN suggests that DNA samples undergo an optional quality control step such as the use of the qBiomarker DNA QC array (SMH-999A). Users then mix genomic DNA with the included qpcr mastermix and then dispense the mixture into every well in the PCR Array. The array is then analyzed in a qPCR thermocycler. For cycling conditions, please refer to the user manual/handbook and the instrument setup guides that are available below. After all of the samples have been analyzed, users then export the CT values from the instrument. The CT values are then analyzed by either the web-based or Excel based data analysis tools that are available.

Sample Guidelines for Mutation Testing
The somatic mutation detection assays and arrays prove to yield accurate and verifiable results in various sample types, including fresh or frozen cell lines, xenografts, tissue samples or fixed samples such as FFPE cell line samples and tissue samples. The amounts listed below are guidelines for starting amounts. If there is less than 200 nanograms of DNA from a fresh or frozen sample then a whole genome amplification step is supported by using Repli-G (Cat. No. 150023 or 150025).

Sample Source Amount of genomic DNA per Array
Fresh or Frozen Cells or Tissue 200 – 500 nanograms
Fixed Cells or Tissue (FFPE) 500- 3000 nanograms

Compatible Instruments
The qBiomarker Somatic Mutation PCR Arrays are compatible with virtually all real-time PCR instruments. The user selects their desired instrument from the table below to identify the correct plate format (plastic plate or ring) with the pre-printed assays. The instrument selection guide below also speciftes the mastermix that is included with the arrays. The only difference between the mastermixes is the inclusion of the instrument-required loading dye.

 

qPCR Instrument Manufacturer Instruments Plate Format Mastermix (included)
QIAGEN Rotor Gene Q 100, Rotor-Gene 6000 (Corbett) R ROX
Agilent / Stratagene Mx3005p, Mx3000p A ROX
Agilent / Stratagene Mx4000 D ROX
Bio-Rad iCycler, iQ5, MyiQ, and MyiQ2 96-well Block A Fluorescein
Bio-Rad Chromo 4 (MJ Research) A ROX
Bio-Rad CFX96, Opticon and Opticon 2 (MJ Research) D ROX
Bio-Rad CFX384 E ROX
Eppendorf ep realplex 2/2S, and 4/4S A ROX
Life Technologies / ABI ABI Standard 5700, 7000, 7300, 7500, 7900HT, ViiA 7 96-well Block A ROX
Life Technologies / ABI ABI 7500 FAST, 7900HT FAST, StepOnePlus, ViiA 7 FAST 96-well Block C ROX
Life Technologies / ABI ABI 7900HT, ViiA 7 384-well Block E ROX
Roche LightCycler 480 96-well Block F ROX
Roche LightCycler 480 384-well Block G ROX
TaKaRa TP-800 A ROX

qBiomarker Somatic Mutation Assays
The qBiomarker Mutation PCR Arrays contain panels of bench-verified hydrolysis probe real-time PCR assays. By combining allele specific amplification and hydrolysis probe detection, QIAGEN has developed real-time PCR assays that detect as low as 1% mutant DNA in a wildtype background. Allele specific amplification is achieved by Amplification Refractory Mutation System (ARMS®) technology, which is based on Taq polymerase discriminating between a match and a mismatch at the 3’ end of the PCR primer (see below). These assays are optimized to work under standard cycling conditions enabling a large number of assays to be analyzed simultaneously.

To ensure assay sensitivity (i.e., the detection of low-percentage mutations) and assay specificity (i.e. the detection of only the mutant allele), we determine for each mutation assay the CT value difference between equal copy numbers of mutant synthetic and wild-type templates (~3000 genome copies, equivalent to 12,000 single-stranded copies). QIAGEN’s assay verification quality control criteria mandate that the mutation assay must have a minimum difference, or detection window, of 8 cycles (see below).

The assay differentiation window (ADW) is defined as the CT difference between the signals generated on a wild-type genomic DNA background and on 100% mutant template by a mutation assay. Assays were verified to have a minimal ADW of 8 but are usually higher. On average the qBiomarker Somatic Mutation Assays have an ADW of 13.6 cycles, allowing for very sensitive detection of low percentage mutations.

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Assay Functional Annotations How It Works References Resources
 

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Assay Functional Annotations How It Works References Resources
 

User Manual qBiomarker Somatic Mutation PCR Array System (PDF)
Data Analysis qBiomarker Somatic Mutation PCR Array Data Analysis Software
Instrument Setup Guides qBiomarker Somatic Mutation PCR Array Instrument Setup Instructions
Application Data Detection Limits, Cancer Pathways
FAQ Frequently Asked Questions about Somatic Mutation Assays and Arrays
Webinar qBiomarker Somatic Mutation Analysis: Real-World Application Data
Slide Presentation> Presentation about qBiomarker Somatic Mutation Assays and Arrays (PDF)
Scientific Poster A Novel Tool for Pathway-Focused Cancer Mutation Profiling (PDF)
Presented at American Association for Cancer Research 2011
White Paper Rapid and accurate cancer somatic mutation profiling with the qBiomarker Somatic Mutation PCR Array (PDF)
Product Profile For screening biology-focused panels of gene mutations (PDF)

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