David Keys

Stem-Loop RT-qPCR for MicroRNA Expression Profiling

Quantification of the microRNAs (miRNAs) in cells or tissues is a crucial step in understanding their biological functions. Development of the stem-loop reverse transcription procedure and TaqMan(®) miRNA assays enables accurate detection of miRNA expression levels by quantitative PCR. Increased experimental throughput permits the expression screening of larger number of miRNAs with small amounts of sample. Here, we demonstrate the use of both TaqMan(®) Array Card and OpenArray(®) platforms to accurately determine the level of miRNA gene expression in biological samples.

Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation.

We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal (‘Culture Adapted’) human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.

TaqMan® Array Cards in Pharmaceutical Research

TaqMan Array Cards are high-throughput, accurate, sensitive, and simple-to-use tools for quantitative analysis of mRNA or miRNA transcripts using a real-time PCR protocol. They utilize a microfluidic card with 384 reaction chambers and eight sample loading ports. For studies of coding transcripts, the reaction chambers are preloaded with user selected or predefined panels of Applied Biosystems TaqMan Gene Expression Assays. These assays enable real-time monitoring of a PCR reaction via hydrolysis of an oligonucleotide probe which has been dual labeled with fluorescent dye and quencher. Applications of TaqMan Array Cards include verification and follow on testing of microarray results, as well as hypothesis driven testing of panels of genes selected for their biological functions and relationships. This chapter describes a protocol for assaying transcription in cultured cells using methods optimized to minimize hands-on time and pipetting steps by skipping RNA isolation and generating cDNA directly in Ambion Cells-to-C(T) lysis solution.

Abstract 4218: Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies.

Somatic mutation has been implicated in many aspects of cancer such as susceptibility, diagnosis, prognosis, drug response and tumor progress. Detection of somatic mutation is of wide interest in cancer research. The rapid advances in next generation sequencing (NGS) technologies have transformed cancer research. For example, the Ion AmpliSeq™ technology enables the selective amplification of 10s to 1000s of target sequences in a single multiplexed PCR and meshes seamlessly with the Ion semiconductor sequencing platform. The Ion Ampliseq™ Comprehensive Cancer Panel (CCP) provides ready-access to hundreds of genes, making it ideal for broad targeted re-sequencing studies. Alternative technologies are in demand to validate the NGS data orthogonally and screen hundreds or more cancer samples to evaluate mutation patterns, prevalence and frequencies in population. Here, we have demonstrated the utility of TaqMan® Mutation Detection Assays using our competitive allele specific TaqMan® PCR (castPCR™) technology in validation of Ion Torrent sequencing data. In this study, we applied the Ion Ampliseq™ Comprehensive Cancer Panel (CCP) panel to NCI-60 cell lines (MCF-7, MDA-MB-231, DU-145, PC-3, SK-MEL- 28) derived from breast, prostate, and skin cancers on Ion Torrent PGM sequencer. We confirmed previously reported mutations in these cell lines and identified the mutations that were not reported before, including missense and non-coding mutations. We then selected a subset of mutation targets from the cancer panel for castPCR validation including the genes of KRAS. EGFR, BRAF, NRAS, PIK3CA, PTEN, KIT, TP53, and more. Since limited sample quantity has been a challenging issue for most cancer researchers, especially for those who are interested in testing multi-targets by qPCR, we developed a preamplification method to enrich the targets of interest prior to running castPCR assays. We compared the Cq values of on-targets and off-targets from preamplified samples and non-preamplified samples, and the data indicates that our preamplification strategy not only provides roughly100 fold target enrichment but also maintains specificity during the preamplification process. For mutation detection, we showed that the mutation data from castPCR™ and from Ion Torrent PGM sequencer share high concordance for any given mutations. The detailed comparison and analysis will be presented and discussed. Our results demonstrate that castPCR™ technology is a valuable validation tool for next generation sequencing. The combination of Ion Torrent sequencing and castPCR validation empowers cancer researchers to understand the roles that somatic mutation plays in cancer. Citation Format: Kelly Li, Cora Woo, Mokang Mouanoutoua, Bonnie Moy, Emanuel Langit, Pius Brzoska, Xiaoqing You, Sejal Desai, David Keys, Junko Stevens, Benjamin Kong, Mark Shannon, Shiaw-Min Chen, David Ruff, Chieh-Yuan Li, David Joun, Iris Casuga, Robert Bennett. Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4218. doi:10.1158/1538-7445.AM2013-4218

Abstract 2100: Cancer biomarker research using castPCR technology

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cancer Biomarkers have applications in the diagnosis, staging, prognosis and monitoring of disease progression, as well as in the predication and monitoring of drug response. Profiling and validation research tools are needed that exhibit the combined features of high sensitivity and high specificity for cancers. However, the sensitivity of molecular methods such as DNA sequencing and conventional genotyping in tumor samples is limited, typically ranging from 5-20%. We have recently developed TaqMan® Mutation Detection Assays using our competitive allele specific TaqMan® PCR (castPCR) technology for cancer biomarker research. castPCR assays were tested with >300 tumor research samples (either fresh/frozen or formalin-fixed, paraffin-embedded samples) and cell lines to assess mutation status. The results showed that castPCR technology can robustly detect mutations as low as 0.1% and has >99% concordance to other technologies including PCR-based technology and sequencing. In this study, a large panel of castPCR assays for AKT1, APC, BRAF, CTNNB1, HRAS, KRAS, NRAS, PIK3CA, PTEN and TP53 genes were used for investigating somatic mutations in breast tumor research samples. Initially, 4 model FFPE cell lines were used to validate the assays. Mutant DNAs were titrated in the wild type DNAs from 50% to 0.1%. Mutations were identified down to 0.1% titration with high reproducibility. No false positives were found in non-tumor samples. The results obtained by castPCR assays for 20 breast tumor samples were concordant to those reported by other methods. Our data showed that castPCR technology provides an excellent tool for identifying cancer biomarkers or confirming potential cancer markers such as those obtained by next-generation sequencing and other technologies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2100. doi:1538-7445.AM2012-2100