Mark Shannon

Expanding applications of protein analysis using proximity ligation and qPCR

The correlation of gene and protein expression changes in biological systems has been hampered by the need for separate sample handling and analysis platforms for nucleic acids and proteins. In contrast to the simple, rapid, and flexible workflow of quantitative PCR (qPCR) methods, which enable characterization of several classes of nucleic acid biomarkers (i.e. DNA, mRNA, and microRNAs), protein analysis methods such as Western blotting are cumbersome, laborious, and much less quantitative. However, TaqMan(R) Protein Assays, which use the proximity ligation assay (PLA) technology, now expand the range of qPCR applications to include the direct detection of proteins through the amplification of a surrogate DNA template after antibody binding. Here we describe an integrated qPCR approach for measuring relative changes in gene and protein expression from the same starting sample and on a single analytical platform that pairs TaqMan Gene Expression (GEx) Assays with TaqMan Protein Assays. We have monitored the changes in mRNA, microRNA, and protein expression of relevant biomarkers in the pluripotent human embryonal carcinoma cell line, NTERA2, upon differentiation to neuronal cells. In addition, TaqMan Protein Assays have been used to monitor protein expression in induced pluripotent stem cells (iPSC) that have been reprogrammed from human somatic cells. The data presented establishes a general paradigm utilizing real-time PCR instruments and reagents for studying the relationship between the stem cell transcriptome and proteome.

Applications of Quantitative Polymerase Chain Reaction Protein Assays During Reprogramming

The capability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) has opened a new area of biology and provides unprecedented access to patient-specific iPSCs for drug screening, disease models, and transplantation therapies. Although the process of obtaining iPSC lines is technically simple, reprogramming is a slow and inefficient process consisting of a largely uncharacterized chain of molecular events. To date, researchers have reported a wide range of reprogramming efficiencies, from <0.01% to >1%, depending on the specific reprogramming factors used, the mode of delivery of the reprogramming factors, properties of the starting cells, and culture conditions. We have applied a quantitative polymerase chain reaction methodology, TaqMan Protein Assays to directly quantify the kinetics, and cellular levels of crucial transcription factors during the reprogramming process. Further, we have used the assays to ascertain the threshold levels of reprogramming protein factors required to generate iPSC colonies, to characterize the protein expression signatures of different iPSC lines, and to rapidly identify iPS versus non-iPSC colonies based on expression of pluripotency markers. These data demonstrate that TaqMan Protein Assays can be used as tools to dissect and gain greater understanding of the mechanisms guiding reprogramming and to further characterize individual established iPSC lines.

Abstract 1163: Sensitive and specific detection of clinically relevant proteins in human testicular germ cell tumors using proximity ligation and quantitative PCR

Human testicular germ cell tumors (TGCTs) are the most frequent solid cancer in Caucasian males between the ages of 15 and 45 years. In spite of the high success rate of current treatment strategies (surgery, irradiation and chemotherapy), TGCTs are the second leading cause of death in this age group. The initiating event, leading to carcinoma in situ (CIS), occurs during embryonal development, offering a window of opportunity for early detection and intervention, thereby preventing the need for intensive (systemic) treatment. CIS is the malignant counterpart of a primordial germ cell, and can be effectively diagnosed using immunohistochemisty for OCT3/4, NANOG, c-KIT and its ligand (stem cell factor, SCF). In addition, SOX17 and SOX2 are informative for the diagnosis of the invasive components seminoma and embryonal carcinoma. Currently, in a clinical setting, these proteins are detected using predominantly immunohistochemistry, an approach that is labor-intensive and non-quantitative. Here we present data on the detection of these proteins using a novel qPCR-based method, Taqman Protein Expression Assay, which is based on the Proximity Ligation Assay (PLA TM ) technology. The method was first validated on negative controls and a series of TGCT-derived cell lines with efficient siRNA-induced down-regulation of the targets, an informative system for investigating stem cell regulatory circuits. Subsequently, the assay was further developed to enable analysis of both snap frozen and formalin-fixed paraffin-embedded (FFPE) tissues. The assays were found to be fast (results in less than 1 day), highly reproducible (SD less than 0.2 C T s), sensitive (able to detect target protein from as few as 10-100 cells, depending on the level of expression of the protein under investigation), and quantitative. Taqman Protein Expression Assays therefore allow the systematic analysis of multiple proteins within a single small sample in a high throughput set up, and offer unique opportunities for sensitive, specific and quantitative protein detection in both experimental and clinical studies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1163.