Project description:To test whether the addition of a peptide nucleic acid (PNA) clamp, which binds WT KRAS at codon 12, can increase the efficacy of mutation detection for KRASG12D within a targeted NGS setting. We tested the effect of clamping the wild-type KRAS sequence in a reference standard (Tru-Q 7, 1.3% Tier from Horizon Diagnostics, Cambridge, UK) with a KRAS c.35G>A mutation (KRASG12D) at an allelic frequency (AF) of 1.3% assessed by digital droplet PCR (ddPCR). We then re-tested the PNA on circulating-free DNA from a patient harbouring a KRASG12D mutation (at an AF of 3.2%, determined by ddPCR). Multiple runs were conducted using 10, 5, 2.5 and 1ng of DNA input.
Project description:Since monozygotic twins (MZTs) share extremely similar genomic DNA sequences, they cannot be distinguished by conventional forensic STR genotyping. The utilization of epigenetic factors in forensic application has gradually risen over the last few years. Epigenetic factors are characterized by dynamic changes and can be either inherited or accumulated as a result of acquired environmental influences. In this study, we analyzed the tsRNAs expression profiles in peripheral blood from four pairs of adult MZTs using PANDORA-sequencing (PANDORA-seq). Subsequently, the differential expressed tsRNAs (DEtsRNAs) were screened and confirmed by RT-qPCR and droplet digital PCR (ddPCR) in both adult and newborn MZTs. Additionally, the characteristics of the DEtsRNAs including longitudinal temporal stability, suitability for aged bloodstains and multigelation were evaluated.
Project description:The current analysis was a two phase study. The first step of the pilot phase comprised an in-silico search for potential target molecules in five different databases. The second step was a screen for potential targets in 28 patients (14 with local relapse and 14 matched controls) by means of microarray technology. The combined results formed the basis for the validation phase in an independent set of 53 patients performed with droplet digital PCR (ddPCR).
Project description:Circular RNA can regulate blood glucose levels by targeting mRNA expression, but the role of circRNA in GDM is still unknown. Therefore, a joint microarray analysis of circRNAs and their targeting mRNAs using the peripheral blood of GDM patients and healthy pregnant women was carried out for the first time. In our study, high-throughput microarray sequencing technique was used to analyze the expression profile of circRNA and transcripts mRNA in the peripheral blood of GDM patients, in order to comprehensively evaluate the role of circRNAs targets and their parents genes in the signal pathways related to the pathogenesis of GDM. Some of the discovered circRNAs and their linear transcript mRNAs related to T cell receptor signaling pathway were further verified in larger samples by droplet digital PCR(ddPCR) and quantitative real-time PCR(qRT-PCR), respectively. The verification results confirmed the initial microarray results.
