| Construction Protocol | pregnancy at 10 weeks gestation, and normal villous tissue from uncomplicated and singleton pregnant women who underwent artificial abortion at 10 weeks gestation. Blood samples (7 mL) were collected before evacuation and placed in tubes containing EDTA. CHM tissue samples after evacuation and normal villous tissue samples after artificial abortion were obtained immediately, and they were placed in RNAlater (Ambion, Austin, TX, USA). Using a mirVana miRNA Isolation Kit (Ambion), total RNA containing small RNA molecules was extracted from each sample immediately after sampling. Quality assessment and concentration measurements of total RNA, including small RNAs, were performed using a Bioanalyzer (Agilent Technologies, South Queensferry, UK) and a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. CHM was diagnosed by histopathological examination and its androgenetic origin (46,XX) was confirmed by DNA genotyping analysis including polymorphic marker of amelogenin gene (AmpFlSTR Identifiler PCR Amplification Kit; Applied Biosystems, Foster City, CA, USA). Normal villous samples were diagnosed by histopathological examination and the biparental origin (46,XX) of CHM was confirmed by the above DNA genotyping analysis. Gestational age for uncomplicated pregnant women was assessed using the crown-rump length (CRL) of fetus measured by ultrasonography, while that for molar pregnancy was decided by the last menstrual period.NGS was applied to a set of CHM tissue and corresponding blood samples from CHM pregnant women at 10 weeks gestation, and normal villous tissue samples from uncomplicated pregnant women at 10 weeks gestation to screen for candidate CHM-pregnancy-associated miRNAs in plasma. Small RNAs were extracted from each sample using a mirVana miRNA Isolation Kit (Ambion) according to the manufacturer?s instructions. RNA quality and the presence of small RNAs were verified on a Bioanalyser (Agilent Technologies). The concentration of RNA was measured on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). After RNA quality was stringently assured, 5 ?g total RNA was used for small RNA library construction using a Small RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer?s instructions. A pair of Illumina proprietary adapters was ligated to the 5? and 3? ends of RNAs, followed by reverse transcription. The small cDNA libraries were amplified by PCR with primers complementary to the adaptor sequences. Subsequently, the libraries were deep sequenced using an Illumina Genome Analyzer II and sequencing data were processed with the Illumina pipeline v1.4, according to the manufacturer?s instructions. |
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