Agarose amplification based sequencing characterization cell-free RNA in preimplantation spent embryo medium.
Anal Chim Acta
; 1296: 342331, 2024 Apr 01.
Article
em En
| MEDLINE
| ID: mdl-38401939
ABSTRACT
BACKGROUND:
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)RESULT:
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 â¼ 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)SIGNIFICANCE:
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).Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Diagnóstico Pré-Implantação
/
Ácidos Nucleicos Livres
Limite:
Female
/
Humans
/
Pregnancy
Idioma:
En
Revista:
Anal Chim Acta
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China
País de publicação:
Holanda