Survival and Treatment Modalities in Primary Vaginal Melanoma-Case Report and a Narrative Review.

Primary vaginal melanoma (PVM) is a rare cancer representing five percent of vaginal cancers and less than one percent of all female vaginal melanomas, with an incidence rate of 0.46 per million women per year. The aim of this study was to present a case of combined therapy and conservative surgical treatment in a young patient with PVM and to perform a systematic review of the same subject.

Topographical transcriptome mapping of the mouse medial ganglionic eminence by spatially resolved RNA-seq.

Background: Cortical interneurons originating from the medial ganglionic eminence, MGE, are among the most diverse cells within the CNS. Different pools of proliferating progenitor cells are thought to exist in the ventricular zone of the MGE, but whether the underlying subventricular and mantle regions of the MGE are spatially patterned has not yet been addressed. Here, we combined laser-capture microdissection and multiplex RNA-sequencing to map the transcriptome of MGE cells at a spatial resolution of 50 μm.

Base preferences in non-templated nucleotide incorporation by MMLV-derived reverse transcriptases.

Reverse transcriptases derived from Moloney Murine Leukemia Virus (MMLV) have an intrinsic terminal transferase activity, which causes the addition of a few non-templated nucleotides at the 3' end of cDNA, with a preference for cytosine. This mechanism can be exploited to make the reverse transcriptase switch template from the RNA molecule to a secondary oligonucleotide during first-strand cDNA synthesis, and thereby to introduce arbitrary barcode or adaptor sequences in the cDNA. Because the mechanism is relatively efficient and occurs in a single reaction, it has recently found use in several protocols for single-cell RNA sequencing.

Quantitative single-cell RNA-seq with unique molecular identifiers.

Single-cell RNA sequencing (RNA-seq) is a powerful tool to reveal cellular heterogeneity, discover new cell types and characterize tumor microevolution. However, losses in cDNA synthesis and bias in cDNA amplification lead to severe quantitative errors. We show that molecular labels--random sequences that label individual molecules--can nearly eliminate amplification noise, and that microfluidic sample preparation and optimized reagents produce a fivefold improvement in mRNA capture efficiency.

Targeted transcript profiling by sequencing.

In this work we present a targeted gene expression strategy employing trinucleotide threading (TnT) amplification and massive parallel sequencing. We have previously shown that TnT combined with array readout accurately monitors expression levels. However, with this detection strategy spurious products go undetected.

Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples.

Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification.

RNA-seq analysis reveals different dynamics of differentiation of human dermis- and adipose-derived stromal stem cells.

Background: Tissue regeneration and recovery in the adult body depends on self-renewal and differentiation of stem and progenitor cells. Mesenchymal stem cells (MSCs) that have the ability to differentiate into various cell types, have been isolated from the stromal fraction of virtually all tissues. However, little is known about the true identity of MSCs.

Highly multiplexed and strand-specific single-cell RNA 5' end sequencing.

Single-cell analysis of gene expression is increasingly important for the analysis of complex tissues, including cancer, developing organs and adult stem cell niches. Here we present a detailed protocol for quantitative gene expression analysis in single cells, by the sequencing of mRNA 5' ends. In all, 96 cells are lysed, and their mRNA is converted to cDNA.

Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq.

Our understanding of the development and maintenance of tissues has been greatly aided by large-scale gene expression analysis. However, tissues are invariably complex, and expression analysis of a tissue confounds the true expression patterns of its constituent cell types. Here we describe a novel strategy to access such complex samples.

Common variants in human CRC genes as low-risk alleles.

The genetic susceptibility to colorectal cancer (CRC) has been estimated to be around 35% and yet high-penetrance germline mutations found so far explain less than 5% of all cases. Much of the remaining variations could be due to the co-inheritance of multiple low penetrant variants. The identification of all the susceptibility alleles could have public health relevance in the near future.

Analysis of short tandem repeats by parallel DNA threading.

The majority of studies employing short tandem repeats (STRs) require investigation of several of these genetic markers. As such, we demonstrate the feasibility of the trinucleotide threading (TnT) approach for scalable analysis of STRs. The TnT method represents a parallel amplification alternative that addresses the obstacles associated with multiplex PCR.

Expression profiling of signature gene sets with trinucleotide threading.

In recent years, studies have shown that expression profiling of carefully chosen intermediary gene sets, comprising approximately 10 to 100 genes, can convey the most relevant information compared to much more complex whole-genome studies. In this paper, we present a novel method suitable for expression profiling of moderate gene sets in a large number of samples. The assay implements the parallel amplification features of the trinucleotide threading technique (TnT), which encompasses linear transcript-based DNA thread formation in conjunction with exponential multiplexed thread amplification.

Allelotyping by massively parallel pyrosequencing of SNP-carrying trinucleotide threads.

Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing.