基于RNA-seq技术筛选低钙培养奶牛乳腺上皮细胞差异表达基因

Screening Differentially Expressed Genes Based on RNA-seq Technology in Cow Mammary Epithelial Cells Cultured with Low Calcium Medium

本研究拟利用转录组测序(RNA-seq)技术筛选低钙培养奶牛乳腺上皮细胞(CMEC)差异表达基因,为临床寻找更为准确、灵敏的奶牛低钙血症生物标志物提供方法与思路。利用RNA-seq技术对低钙组CMEC转录组测序,筛选8个差异表达基因进行定量反转录聚合酶连锁反应(qRT-PCR)验证测序结果准确性,对测序数据进行基因功能注释及通路富集分析。结果显示,2组分别得到63910842条和66979098条滤过读段,共计筛选116个差异表达显著性基因,其中上调基因52个,下调基因64个,并找到调节甲状腺素信号通路的KAT2A基因和参与甲状腺素合成的DUOXA2基因。利用qRT-PCR对测序结果中筛选的8个差异表达基因进行验证,其结果与RNA-seq结果大体一致。结果表明低钙环境影响CMEC基因表达,KAT2A和DUOXA2基因可能参与细胞钙调节。

In order to provide methods and ideas for finding more accurate and sensitive biomarkers of hypocalcemia in periparturient cows.Differentially expressed genes in low-calcium-cultured cow mammary epithelial cells(CMEC)were screened by RNA-seq technology.Transcriptome of the cells in the control group and the low-calcium group was sequenced by RNA-seq technology.Eight differentially expressed genes were selected for qRT-PCR to verify the accuracy of sequencing results.Gene function annotation and pathway enrichment analysis were performed on sequencing data.Transcriptome sequencing in the two group of cells obtained 63910842 and 66979098 clean sequences,respectively.A total of 116 differentially expressed genes were screened,of which 52 were up-regulate and 64 were down-regulated.The KAT2A gene that regulates the thyroxine signaling pathway and the DUOXA2 gene involved in thyroxine synthesis were found.The 8 differentially expressed genes screened in the sequencing results were verified by qRT-PCR.The results were found to be roughly consistent with the RNA-seq results.The low-calcium culture environment affects the gene expressions in CMEC.The KAT2A and the DUOXA2 genes may be involved in calcium regulation of the cells.