Claudia Litterst

Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase.

Using an automated cell counter to simplify gene expression studies: siRNA knockdown of IL-4 dependent gene expression in Namalwa cells.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression. siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks. These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology. Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages. To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis. The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependant gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa). The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells. We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

Very Low Abundance Single-Cell Transcript Quantification with 5-Plex ddPCR TM Assays

Gene expression studies have provided one of the most accessible windows for understanding the molecular basis of cell and tissue phenotypes and how these change in response to stimuli. Current PCR-based and next generation sequencing methods offer great versatility in allowing the focused study of the roles of small numbers of genes or comprehensive profiling of the entire transcriptome of a sample at one time. Marrying of these approaches to various cell sorting technologies has recently enabled the profiling of expression in single cells, thereby increasing the resolution and sensitivity and strengthening the inferences from observed expression levels and changes. This chapter presents a quick and efficient 1-day workflow for sorting single cells with a small laboratory cell-sorter followed by an ultrahigh sensitivity, multiplexed digital PCR method for quantitative tracking of changes in 5-10 genes per single cell.

Abstract 2301: Cross validation of NGS methylated targets using droplet digital PCR (ddPCR)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA methylation is one of the most studied epigenetic modifications. DNA methylation plays an important role in a number of physiological processes as well as common diseases such as cancer and neurodegenerative disorders. In mammals, DNA methylation occurs at the C-5 position of cytosine in CpG dinucle¬otide sequences, which are mainly concentrated in regions known as CpG islands. Methylation in CpG islands within gene promoters usually leads to gene silencing. Recent data have shown the correlation of methylation and disease status. However, there are significant limitations in both biology and technology. Biologically, methylation status is highly heterogeneous in nature. In normal controls there is never a complete lack of methylation marks, and conversely, never a completely saturated methylation signature. In addition methylation sites of interest are often located in CpG islands, regions where multiple methylation sites occur side by side, making assay design and discrimination challenging. Technological hurdles add significant variability as well. The typical method for determining methylation is bisulfite conversion, which uses harsh chemicals to convert cytosines to uracil. When a cytosine is methylated, it is largely, though not completely, protected from the conversion process. However, bisulfite conversion also causes a great deal of DNA damage, causing the strands to separate and fragment. In most cases of research, the DNAs that researchers want to examine are from FFPE or cfDNA samples, samples which are already both limited and damaged. Because of these challenges, there is an unmet need for a sensitive yet robust method to analyze DNA methylation in clinical samples in a rapid, inexpensive, and high throughput manner. Herein we showcase the use of ddPCR for detection of DNA methylation in bisulfite converted gDNA samples. First we demonstrate the superior sensitivity, linearity and robustness of ddPCR methylation detection in the SNRPN promoter, a model system for an imprinted gene involved in neurological disorders. Secondly we show the detection of DNA methylation in the vimentin promoter, which is known to be methylated in cancer (Li et al. Nat. Biotech. 2009). We will demonstrate reproducible detection in replicate bisulfite converted samples and sensitivity. Citation Format: Dawne N. Shelton, Claudia Litterst, John F. Regan, Helen R. Moinova, Sanford D. Markowitz. Cross validation of NGS methylated targets using droplet digital PCR (ddPCR). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2014-2301