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FAQs : Mutation PCR Arrays & Assays

Real-time PCR and PCR Array Technical FAQ

Q:What is the sensitivity of the assays contained on the somatic mutation PCR arrays?
A:The assays were validated to have at least 1% sensitivity (i.e. able to detect 1% mutation on a wildtype background), but the sensitivity is usually higher. On average, the assay sensitivity is 0.03%.

Q:How many mutations can I profile per sample on the somatic mutation PCR array?
A:Depending on the array that is chosen, one can profile between 40 and 360 mutations per sample in one PCR run.

Q:Where can I obtain the complete mutation assay list?

Q:Were all assays on the arrays bench-validated?
A:Each array contains a panel of assays bench-validated for hydrolysis probe based real-time RT-PCR detection. These assays are optimized to work under standard cycling conditions enabling a large number of assays to be analyzed simultaneously.

Q:How do you decide on which assays to develop? How well do they cover the somatic mutations published so far in the literature?
A:For assay development, our strategy was to comprehensively cover the most frequently detected, functionally significant cancer mutations in public databases. Currently our ~800 assay collection covers 75% of all somatic mutation occurrences reported in the literature.

Q:How do you decide on the assays to include on the pathway arrays and disease arrays?
A:For pathway-focused arrays, we included assays for detecting the most frequent and functionally verified mutations for multiple genes within a specific pathway implicated in a variety of cancers. Additional assays are also available for each gene to allow array customization. For disease-focused arrays, we selected top somatic mutations for that disease type covering between 4,000 and 40,000 published tumor samples for each disease type.

Q:What are the control features on the arrays?
A:Each array contains gene copy reference assays for each gene represented by the array. These assays target non-variable regions of the genes and measure input DNA quality and amount. In addition, these assays sensitively measure gene dosage to normalize mutation assay data against the gene copy number. Each array also contains positive PCR controls (SMPC) to test for the presence of inhibitors in the sample or the efficiency of the polymerase chain reaction itself using a pre-dispensed artificial DNA sequence and the primer set that detects it.

Q:What is the data analysis method, and where can I find it?
A:The choice between one of two data analysis methods depends on the experimental setup and sample type.
  1. ΔΔCt Method

  2. Recommended for experiments using:
    Small (four or less) number of fresh, frozen samples
    Large number of samples with similar DNA quality
  3. Average Ct Method

  4. Recommended for experiments using:
    FPPE samples, large number of samples or without wild-type control samples
Data analysis can either be performed on the somatic mutation data analysis web portal or by downloading the Excel data analysis templates at http://www.sabiosciences.com/somaticmutationdataanalysis.php

Q:What is the principle for somatic mutation PCR array data analysis?
A:The basic principle behind the data analysis is that we compare the Ct value of a mutation assay in a test sample with the Ct value of the same assay in a wildtype sample. When there is a significant difference (a preset value of 4 Cts) between the Ct values, the test sample is concluded to contain the mutation. The Ct values used for comparison can either be raw Ct (in average Ct method) or normalized Ct (in delta delta Ct case). When the Ct difference falls between 3 and 4, we give a borderline mutation call, which means that the mutation may be present at low percentage. When the Ct difference is smaller than 3, we give a negative mutation call (i.e. the mutation percentage is beyond the detection limit of the array). The wildtype sample can be either a genuine wildtype sample that is tested in the same experiment, or it could be a "virtual" wildtype sample that is computed from all test samples. For detailed description of the data analysis principle, refer to (link to white paper).

Q:What PCR machine do I need to use to run such an array?
A:The arrays are designed for routine use on any PCR instruments. Arrays are available in the following formats:
qBiomarker Somatic Mutation PCR Array Format A: Fluoroscein, 96-well; for Bio-Rad
iCycler, iQ5, MyiQ, and MyiQ2 instruments
qBiomarker Somatic Mutation PCR Array Format A: ROX, 96-well; for ABI Standard
96-well Blocks (5700, 7000, 7300, 7500, 7900HT, ViiA 7); Bio-Rad Chromo 4 (MJ
Research); Stratagene Mx3005p, Mx3000p; Eppendorf ep realplex 2/2S, and 4/4S
instruments
qBiomarker Somatic Mutation PCR Array Format C: ROX, 96-well; for ABI 7500 FAST
96-well Block, 7900HT FAST 96-Well Block, StepOnePlus, and ViiA 7 FAST 96-well Block
instruments
qBiomarker Somatic Mutation PCR Array Format D: ROX, 96-well; for Bio-Rad CFX96,
Opticon and Opticon 2 (MJ Research); Stratagene Mx4000 instruments
qBiomarker Somatic Mutation PCR Array Format E: ROX, 384-well; for ABI 7900HT
384-well Block, ViiA 7 384-well Block; Bio-Rad CFX384 instruments
qBiomarker Somatic Mutation PCR Array Format F: ROX, 96-well; for Roche
LightCycler 480 96-well Block instruments
qBiomarker Somatic Mutation PCR Array Format G : ROX, 384-well; for Roche
LightCycler 480 96-well Block instruments

Q:What effects can be predicted with poor sample quality?  How robust is the platform with questionable sample quality (such as FFPE samples)?
A:Poor quality samples tend to give higher Cts in all assays (mutation assays and gene copy number assays) and there are two possible consequences: (1) if using average Ct method for data analysis, even real mutations in poor quality samples will not be called, because the mutation locus Ct will be pushed to a high Ct region; (2) if using delta delta Ct method for data analysis, there will be a number of false positives in low quality samples.

We recommend using the average Ct value for gene copy number assays on the array to gauge the sample quality (or run the sample on a DNA QC plate before running samples on an array). For FFPE samples, we recommend the average Ct to be below 32 to allow sensitive detection of mutations. Samples that meet this criterion perform robustly on the arrays.

Q: What sample types can I test on the arrays?
A:The somatic mutation detection assays and arrays prove to yield accurate and verifiable results in various sample types, including fresh frozen cell lines and tissue samples, cell line admixtures, FFPE cell line samples and FFPE tissue samples from various sources.

Q: What method(s) do you recommend as an alternative way to validate the mutations identified on the somatic mutation PCR arrays?
A:Sanger sequencing and pyrosequencing can be used to validate the mutations identified on the arrays. However, one needs to bear in mind that the detection sensitivity of Sanger sequencing is around 20%, and the detection sensitivity of pyrosequencing is around 5%, while RT-PCR based arrays can detect 1% or lower mutations. Therefore, mutations occurring below the detection limit of Sanger sequencing and pyrosequencing will not be verified by these two methods.

Q: What are the advantages of RT-PCR based somatic mutation PCR arrays and assays compared to other platforms?
A:The main advantages are qPCR-based superior detection sensitivity and straightforward data analysis procedure. Additional major advantages over other currently available mutation detection platforms/methods are: (1) The workflow is very simple, involving only one setup step. No multi-step handling is involved, and hands-on time is less than any other method available. (2) Reactions involved are all closed-tube reactions avoiding sample contamination. (3) The DNA sample input is low. (4) The hardware involved in analysis using the mutation detection arrays and assays is highly accessible, enabling such analysis for any laboratory with access to real-time PCR instruments.

Q: What pathways and diseases are currently covered?
A:The pathways covered include major receptor tyrosine kinase pathways, non-receptor kinase pathways, as well as additional oncogene and tumor suppressor pathways; and the targeted diseases include all major cancer types. In addition, a collection of more than 800 pre-validated somatic mutation assays enables researchers to study single mutations or to customize the mutation panels or collections according to their research needs.

Q: How do I use the arrays?
A:The simple workflow involves mixing the DNA sample of interest with ready-to-use qBiomarker Probe Master Mix, aliquoting the mixture into the array plate wells, performing real-time PCR, and making mutation/genotype calls using web-based data analysis software or Excel-based templates.

Q:What are the technological principles behind somatic mutation PCR arrays and assays?
A:Real-time PCR is the most sensitive and reliable method for the detection of DNA mutations. By combining allele specific amplification and hydrolysis probe detection, we have developed real-time PCR assays that detects as low as 1% somatic mutations in the background of wild-type genomic DNA. Allele specific amplification is achieved by Amplification Refractory Mutation System (ARMS@) technology, which is based on the ability of Taq polymerase to discriminate between a match and a mismatch at the 3' end of the PCR primer.