RNA Sequencing Facilitates Quantitative Analysis of differentially expressed genes after UCA1 knockdown in human erythroid cells. RNA Sequencing Facilitates Quantitative Analysis of differentially expressed genes after UCA1 knockdown in human erythroid cells

Purpose: The goals of this study are to determine possible downstream targets of UCA1 on day8 of erythroid differentiation with or without UCA1 knockdown. Methods:gene expression profiling with or without UCA1 knockdown in differentiated erythroblasts at day8 were generated by deep sequencing, induplicate, using Illumina GAIIx.The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Conclusions: heme metabolism genes were down-regulated after UCA1 knockdown during erythroid differentiation Overall design: mRNA profiles of day8 erythroid cells with or without UCA1 knockdown were generated by deep sequencing, in duplicate, using Illumina GAIIx.

研究目的：本研究旨在明确红系分化第8天时，敲低UCA1与未敲低UCA1情况下的潜在下游靶基因。
实验方法：采用Illumina GAIIx测序平台，对第8天分化的红系母细胞中敲低与未敲低UCA1的样本进行高通量测序，设置双重复。对通过质量过滤的序列读段，在转录本异构体水平采用两种分析方法：先使用Burrows-Wheeler比对工具（Burrows–Wheeler Aligner，BWA）结合方差分析（ANOVA），另一种为TopHat结合Cufflinks。随后采用TaqMan探针法与SYBR Green染料法进行qRT-PCR验证。
研究结论：红系分化过程中敲低UCA1后，血红素代谢相关基因表达量下调。
整体实验设计：采用Illumina GAIIx测序平台，对第8天的红系细胞中敲低与未敲低UCA1的样本进行高通量测序，设置双重复，获取mRNA表达谱。