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
Long noncoding RNAs ( lncRNAs ) are transcribed RNA molecules greater than 200 nucleotides in length. In recent years, studies have shown that lncRNAs have many important bi-ological functions. With the development of modern biological technology, lncRNAs transcripts can be easily detected and pre-dicted. However, unlike protein-coding genes whose sequence motifs usually have indicative function, lncRNA sequence infor-mation is currently uninformative for function prediction. Thus, how to decode the function of lncRNAs has gradually become a hot issue in genome research. The paper summarizes the studies regarding the methods to probe the function of lncRNAs.
ABSTRACT
Objective To construct a recombinant plasmid carrying phoS2 from Mycobacterium tuberculosis, to express prokaryotically,to purify the recombinant protein and to identify the immunogenicity of its recombinant protein.Methods phoS2 gene was subcloned into expression vector pET32a.The constructed recombinant plasmid phoS2/pET32a was transformed to E.coli BL21 (DE3)for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and then purified by His-bind affinity chromatography with Ni2+.Results of the induced expression level were detected by SDS-PAGE. The recombinant protein was purified by affinity chromatography.The biological activity of phoS2 was tested by Western blot;its secondary structure and 3-D structure were predicted with software packages such as DNAstar and Rasmol.Results The recombinant plasmid phoS2/pET32a of Mycobacterium tuberculosis was constructed successfully and confirmed by restriction endonuclease analysis and DNA sequencing analysis. SDS-PAGE analysis showed that the expressed proteins were mainly insoluble;Western blot analysis showed that the molecular weight of phoS2 recombinants protein was 3 7 9 5 3 u.The recombinant protein was purified by affinity chromatography.Analysis of the predicted protein indicated that the molecular mass was 37 953.1 ku,PI was 5.75,there was one transmembrane region and function sites were 1 to 370. Conclusion The recombinant plasmid phoS2/pET32a of Mycobacterium tuberculosis was successfully constructed and phoS2 gene could be expressed in BL2 1 with high efficiency.Predicting protein structure and function can provide some theoretical basis for conducting the present experiment and selecting further research.