Project description:Introduction: This study investigates the active component Mannose-B from Codonopsis pilosula and its effect on human trophoblast cell function, particularly focusing on the regulation of Laminin Subunit Beta 1 (LAMB1) expression and its implications in subchorionic hematoma (SCH). Methods: Key genes involved in SCH pathology were identified through RNA sequencing and bioinformatics analysis. Network pharmacology was utilized to screen active components in Codonopsis pilosula and their critical targets. In vitro, HTR-8/Svneo cells were used to assess proliferation, migration, and invasion through CCK8, Transwell, and cell migration assays. A SCH rat model was established to evaluate changes in coagulation parameters, litter size, fetal viability, and fetal and placental weights. In vivo validation of Mannose-B's effects on LAMB1 expression and SCH pathology was performed using RT-qPCR and Western Blot. Results: Network pharmacology and molecular docking identified Mannose-B as an effective compound in Codonopsis pilosula, potentially beneficial for SCH treatment, with LAMB1 as a significant target. In vitro experiments showed that Mannose-B enhanced HTR-8/Svneo cell proliferation, migration, and invasion by reducing LAMB1 expression. In vivo experiments confirmed Mannose-B's inhibitory effect on placental LAMB1 expression and its potential in ameliorating SCH pathology. Conclusion: Mannose-B from Codonopsis pilosula inhibits LAMB1 expression, promoting human placental trophoblast cell proliferation, migration, and invasion, thereby mitigating the progression of SCH pathology.
Project description:Gene expression profiles generated with RNA sequencing can be biased by RNA amount and methods utilised for cDNA library generation. Polymerase chain reaction (PCR) amplification can generate high PCR duplicate proportions and introduce bias in transcript counts. In this study, we investigate the impact of input amount and PCR cycle number on the PCR duplication rate and on the RNA-seq data quality. We used a range of inputs (1 ng - 1,000 ng) and assessed the PCR duplication rate using unique molecular identifiers (UMIs). For broader applicability, we sequenced the data on four different short-read sequencing platforms: Illumina NovaSeq 6000, Illumina NovaSeq X, Element Biosciences AVITI, and Singular Genomics G4. We highlight the limitations of using input amounts below 125 ng and the advantages for using UMIs for deduplication. We contrast the data obtained from different sequencers and discuss the benefits and drawbacks of using Illumina library conversion kits.