Exploring the impact of primer length on efficient gene detection via high-throughput sequencing.

Reverse transcription (RT) is a crucial step in most RNA analysis methods. Optimizing protocols for this initial stage is critical for effective target detection, particularly when working with limited input RNA. Several factors, such as the input material quality and reaction conditions, influence RT efficiency. However, the effect of RT primer length on gene detection efficiency remains largely unknown. Thus, we investigate its impact by generating RNA-seq libraries with random RT primers of 6, 12, 18, or 24 nucleotides. To our surprise, the 18mer primer shows superior efficiency in overall transcript detection compared to the commonly used 6mer primer, especially in detecting longer RNA transcripts in complex human tissue samples. This study highlights the critical role of primer length in RT efficiency, which has significant potential to benefit various transcriptomic assays, from basic research to clinical diagnostics, given the central role of RT in RNA-related analyses.

RIPK3 promoter hypermethylation in hepatocytes protects from bile acid-induced inflammation and necroptosis.

Necroptosis facilitates cell death in a controlled manner and is employed by many cell types following injury. It plays a significant role in various liver diseases, albeit the cell-type-specific regulation of necroptosis in the liver and especially hepatocytes, has not yet been conceptualized. We demonstrate that DNA methylation suppresses RIPK3 expression in human hepatocytes and HepG2 cells. In diseases leading to cholestasis, the RIPK3 expression is induced in mice and humans in a cell-type-specific manner. Overexpression of RIPK3 in HepG2 cells leads to RIPK3 activation by phosphorylation and cell death, further modulated by different bile acids. Additionally, bile acids and RIPK3 activation further facilitate JNK phosphorylation, IL-8 expression, and its release. This suggests that hepatocytes suppress RIPK3 expression to protect themselves from necroptosis and cytokine release induced by bile acid and RIPK3. In chronic liver diseases associated with cholestasis, induction of RIPK3 expression may be an early event signaling danger and repair through releasing IL-8.

Characterization of RNA content in individual phase-separated coacervate microdroplets.

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

Advances in Non-Coding RNA Sequencing.

Non-coding RNAs (ncRNAs) comprise a set of abundant and functionally diverse RNA molecules. Since the discovery of the first ncRNA in the 1960s, ncRNAs have been shown to be involved in nearly all steps of the central dogma of molecular biology. In recent years, the pace of discovery of novel ncRNAs and their cellular roles has been greatly accelerated by high-throughput sequencing. Advances in sequencing technology, library preparation protocols as well as computational biology helped to greatly expand our knowledge of which ncRNAs exist throughout the kingdoms of life. Moreover, RNA sequencing revealed crucial roles of many ncRNAs in human health and disease. In this review, we discuss the most recent methodological advancements in the rapidly evolving field of high-throughput sequencing and how it has greatly expanded our understanding of ncRNA biology across a large number of different organisms.