Next Generation Sequencing Facilitates Transcriptomes Quantitative Analysis of siRNA mediated ASGR1 knockdown

Purpose: The goal of this study is to demonstrate the gene profiles of ASGR1 knock down mediated by siRNA compared with siControl in Huh7 cells. Methods: The Huh7 cells were transfected with small interference RNA (siRNA) targeting scramble (WT) or ASGR1 (AS2) for 8 hours, then cells were refreshed with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 100 units ml-1 penicillin, 100 μg ml-1 streptomycin sulfate, and 10% fetal bovine serum (FBS). After 72 hours, the cells were harvested for RNA isolation and followed by high-throughput sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcriptome with Hisat2(v2.0.1). qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 45 million sequence reads per sample to the human genome in the WT and AS2 cell lines Hisat2 (v2.0.1). RNA-seq data confirmed stable expression of the known housekeeping genes, and these genes were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than a goodness of fit (R2) of 0.9. Approximately 4.2% of the transcripts showed significantly differential expression between the WT and AS2 cell lines, with a log2 fold change ≥1.0 and p value <0.05. Altered expression of 5 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed transcriptomes analysis of ASGR1 knock down in Huh7 cells, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within the WT and AS2 cell. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. transcriptome profiles of siRNA targeting scramble (WT) or ASGR1 (AS2) for 72 hours in Huh7 cells.