Single-molecule RNA-FISH analysis reveals stochasticity in reactivation of latent HIV-1 regulated by Nuclear Orphan Receptors NR4A and cMYC.

HIV-1 eradication strategies require complete reactivation of HIV-1 latent cells by Latency Reversing Agents (LRA). Current methods lack effectiveness due to incomplete proviral reactivation. We employed a single-molecule RNA-FISH (smRNA-FISH) and FISH-Quant analysis and found that proviral reactivation is highly variable from cell-to-cell, stochastic, and occurs in bursts and waves, with different kinetics in response to diverse LRAs.

Generic Multiplex Digital PCR for Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples.

An accurate T cell quantification is prognostically and therapeutically relevant in various clinical applications, including oncology care and research. In this chapter, we describe how T cell quantifications can be obtained from bulk DNA samples with a multiplex digital PCR experiment. The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: a unique T cell DNA marker, a regional corrector, and a reference DNA marker.

Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction.

Assessment of specific β-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for isolation. Recently, several groups have demonstrated that unmethylated insulin ()-DNA is correlated to β-cell death in type 1 diabetes patients and during clinical islet isolation and subsequent transplantation. Here, we present a step-by-step protocol of our novel developed method for quantification of the relative amount of unmethylated -DNA using methylation sensitive restriction enzyme digital polymerase chain reaction This method provides a novel and sensitive way to quantify the relative amount of β-cell derived unmethylated -DNA in cellular lysate.

A novel digital PCR-based method to quantify (switched) B cells reveals the extent of allelic involvement in different recombination processes in the IGH locus.

B cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For clinical applications, it can be important to quantify (switched) B cells accurately in a variety of body fluids and tissues of benign, inflammatory and malignant origin.

Scientific and clinical implications of genetic and cellular heterogeneity in uveal melanoma.

Here, we discuss the presence and roles of heterogeneity in the development of uveal melanoma. Both genetic and cellular heterogeneity are considered, as their presence became undeniable due to single cell approaches that have recently been used in uveal melanoma analysis. However, the presence of precursor clones and immune infiltrate in uveal melanoma have been described as being part of the tumour already decades ago.