Genechip command console software version 4

For analysis of the miRNA array, CEL-files of the raw data were produced with Affymetrix GeneChip Command Console Software Version 4.0. (Affymetrix, Santa Clara, CA, U.S.) Partek Genomics Suite software (Version 6.14.0923; Partek, Inc., St. Louis, MO, USA) was used for further analysis. CEL-files were imported including control and interrogating probes, and the arrays were normalized using quantile normalization. Probeset summarization was done using Median Polish. Probe values were log2 transformed. In order to detect differential miRNA expression between the 2 groups, 1-way ANOVA was performed [35 (link)] and Fisher’s Least Significant Difference (LSD) was used as contrast method.
MultiExperiment Viewer 4.8 was used for hierarchical clustering of miRNA array data [36 (link)]. 2^-ΔΔCt and patients’ demographic data and blood counts were compared using Student’s t-test for independent samples. p-values were rounded to 4 significant digits; p-values below 0.05 were considered statistically significant.

RNA from primary adult NSPCs for microarray analysis was isolated with the RNeasy Micro Kit (Qiagen), according to the manufacturer’s protocol. RNA samples were further processed by the Ambion WT Expression kit as described by the manufacturer (Life Technologies). Fragmentation and labeling of the amplified cDNAs was done with the GeneChip WT Terminal Labeling and Controls Kit (Affymetrix). Labeled fragments were hybridized to Affymetrix GeneChip ST 1.0 arrays for 16 h at 45 °C with 60 rpm in an Affymetrix Hybridization oven 645. After washing and staining, the arrays were scanned with the Affymetrix GeneChip Scanner 3000 7 G. CEL files were produced from the raw data with Affymetrix GeneChip Command Console Software Version 4.0. Partek Genomics Suite software (Version 6.13.0412) was employed for further analyses. CEL files were imported, including control and interrogating probes, pre-background adjustment was set to adjust for GC content and probe sequence, and RMA background correction was performed. Arrays were normalized using quantile normalization, and probe-set summarization was done using median polish. Probe values were log2 transformed. To identify differentially expressed genes between the groups, we performed a one-way ANOVA in Partek.

Individuals who showed abnormalities in NIPT received ultrasound‐guided amniocentesis at 18‐24 weeks of pregnancy after informed consent was signed by pregnant women and their families. Amniotic fluid samples were collected from the patients for conventional G‐banded cytogenetic assays and microarray analysis. Amniocytes were isolated and cultured using BIO‐AMF™‐2 medium (Biological Industries, Kibbutz Beit‐Haemek, Israel) and Chang Medium^® D (Irvine Scientific, Santa Ana, CA, USA) at 37°C with 5% CO₂ for 6‐7 days. The cells at metakinesis were harvested to prepare slides according to the statements in published article.¹⁶ Then, G‐band staining was performed according to the Internal System for Human Cytogenomic Nomenclature 2016.
In addition, microarray analysis was also performed for the patients. In brief, genomic DNA was extracted from amniotic fluids using Genomic DNA Extraction kit (QIAamp DNA Blood Mini kit; Qiagen GmBH, Hilden, Germany). DNA samples were digested, ligated and amplified via PCR method. Then, obtained products were processed according to standard procedures of Affymetrix CytoScan 750K Array analysis. The results were processed in Affymetrix GeneChip Command Console software (version 4.0) and Chromosome analysis software (Chromosome Analysis Suite version 2.1) (Affymetrix; Thermo Fisher Scientific, Inc).