ABSTRACT
Objective : To verify the feasibility of replacing the expensive commercial reagent SMART-Seq v4 Ultra Low Input RNA Kit (hereinafter referred to as TaKaRa reagent) with a reagent (hereinafter referred to as DIY reagent) which was made by ourselves based on the SMART (switching mechanism at 5' end of RNA template) technology. Methods ¡¤ Four 8-week-old C57BL/6 female mice were randomly di-vided into two groups. One group did not receive any treatment as a control, and the other group was intraperitoneally injected with 1 mL of 4% thioglycollate broth to induce peritoneal macrophages. After 72 hours, RNA was extracted from the peritoneal macrophages. cDNA library con-struction was performed with DIY reagent and TaKaRa reagent respectively. Finally, bioinformatics analysis was performed to compare the RNA sequencing results after use of different library construction reagents from different aspects, such as data quality, gene differential expression analysis, and KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. Results ¡¤ The results of bioinformatics analysis showed that the sample processed by the DIY reagent and TaKaRa reagent were both of good data quality, and the two reagents had comparative capabil-ity in transcripts capture. Gene coverage of the sequences both showed consistent uniformity. On top of these, the results of differential gene ex-pression analysis and gene pathway analysis were consistent. Conclusion ¡¤ Considering relatively great reduction in experimental cost for li-brary construction, the DIY reagent can replace expensive commercial reagent for library construction experiments with a small amount of cell input.
ABSTRACT
Objective: In order to explore the expression of sinomenine content control genes, synthetic control sites and expression pathway. Methods: In this study, high performance liquid chromatography (HPLC) was used to determine the content of sinomenine in the roots and stems of 49 Sinomenii Caulis in six populations. Two populations with large multiple differences in sinomenine content were selected, namely Shanxi Baoji and Guizhou Zunyi. The most representative of them were selected, and their roots and stems were taken for transcriptome sequencing and named as HR/LR and HS/LS. Results: Sequencing results showed that 355 201 transcripts were obtained by splicing clean reads, including 275 491 Unigene transcripts. There were 23 562 and 37 143 differentially expressed genes in HR/LR and HS/LS, respectively. GO database analysis showed that the functions of these differentially expressed genes were significantly enriched in aspartic-type endopeptidase activity and aspartic-type peptidase activity, it is speculated that these two enzymes might be encoded. The results of KEGG enrichment explained that the differentially expressed genes were involved in carbohydrate metabolism, protein binding to cell membrane and vitamin C synthesis. The results of qRT-PCR verified the expression of upstream key genes of the isoquinoline alkaloid synthesis pathway and found that it was positively correlated with the accumulation of sinomenine. Conclusion: This study provided a preliminary understanding of the molecular mechanism that caused the difference in sinomenine content, and provided a reference for further understanding of the accumulation rules and synthesis pathways of sinomenine.