Agarose amplification based sequencing characterization cell-free RNA in preimplantation spent embryo medium

化学 胚泡 核糖核酸 胚胎 琼脂糖 分子生物学 RNA提取 细胞生物学 色谱法 生物化学 基因 胚胎发生 生物
作者
Huajuan Shi,Qinyu Ge,Min Pan,Yuqi Sheng,Ting Qi,Ying Zhou,Yuqing Sun,Yunfei Bai,Lingbo Cai
出处
期刊:Analytica Chimica Acta [Elsevier BV]
卷期号:1296: 342331-342331
标识
DOI:10.1016/j.aca.2024.342331
摘要

The cell-free RNA (cf-RNA) of spent embryo medium (SEM) has aroused a concern of academic and clinical researchers for its potential use in non-invasive embryo screening. However, comprehensive characterization of cf-RNA from SEM still presents significant technical challenges, primarily due to the limited volume of SEM. Hence, there is urgently need to a small input liquid volume and ultralow amount of cf-RNA library preparation method to unbiased cf-RNA sequencing from SEM. (75) Here, we report a high sensitivity agarose amplification-based cf-RNA sequencing method (SEM-Acf) for human preimplantation SEM cf-RNA analysis. It is a cf-RNA sequencing library preparation method by adding agarose amplification. The agarose amplification sensitivity (0.005 pg) and efficiency (105.35 %) were increased than that of without agarose addition (0.45 pg and 96.06 %) by ∼ 90 fold and 9.29 %, respectively. Compared with SMART sequencing (SMART-seq), the correlation of gene expression was stronger in different SEM samples by using SEM-Acf. The cf-RNA number of detected and coverage uniformity of 3′ end were significantly increased. The proportion of 5′ end adenine, alternative splicing events and short fragments (<400 bp) were increased. It is also found that 4‐mer end motifs of cf-RNA fragments was significantly differences between different embryonic stage by day3 spent cleavage medium and day5/6 spent blastocyst medium. (141) This study established an efficient SEM amplification and library preparation method. Additionally, we successfully described the characterizations of SEM cf-RNA in preimplantation embryo using SEM-Acf, including expression features and fragment lengths. SEM-Acf facilitates the exploration of cf-RNA as a noninvasive embryo screening biomarker, and opens up potential clinical utilities of small input liquid volume and ultralow amount cf-RNA sequencing. (59).
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