cell type: Human amniotic fluid stem cells (hAFSC) time point: after 7-day osteogenic induction
Treatment protocol
The hAECs, hBMSCs and hAFMSCs were cultured in a classical osteogenic induction medium [α-MEM (Invitrogen, China) supplemented with 10% FBS (Gibico, China), 0.1 mM ascorbic acid (Sigma-Aldrich, USA), 10 mM β-glycerophosphate (Sigma-Aldrich, USA), 10-5 mM dexamethasone (Sigma-Aldrich, USA)]. After the 7-day osteogenic induction, total RNA was extracted with RNAiso Plus reagent (Takara, Japan).
Growth protocol
The hAECs, hBMSCs and hAFMSCs were cultured in the expansion culture medium (ECM) with regular passaging. The ECM for hAECs was prepared with DMEM/F12 medium (Invitrogen, China) supplemented with 2 mM L-Glutamine (Invitrogen, China), 10 ng/ml rhEGF (Invitrogen, China), 20% fetal bovine serum (Gibco, China) and 1% antibiotic-antimycotic (Gibco, China). The ECM for hBMSCs and hAFMSCs was prepared with DMEM/F12 medium (Invitrogen, China) supplemented with 2 mM L-Glutamine (Invitrogen, China), 10 ng/ml rhFGF (Invitrogen, China), 20% fetal bovine serum (Gibco, China) and 1% antibiotic-antimycotic (Gibco, China).
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions. Total RNAs was quantified by the NanoDrop ND-2000 (Thermo Scientific) and the RNAs integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies).
Label
biotin
Label protocol
The sample labeling was performed based on the manufacturer’s standard protocols.
Hybridization protocol
The microarray hybridization was performed based on the manufacturer’s standard protocols.
Scan protocol
The arrays were scanned by the Affymetrix Scanner 3000(Affymetrix)
Description
AFSC D7
Data processing
Affymetrix GeneChip Command Console (version 4.0, Affymetrix) software was used to extract raw data. Next, Expression Console (version1.3.1, Affymetrix) software offered RMA normalization for both gene and exon level analysis. Then the gene expression analysis and alternative splice analysis proceeded separately. [1] Gene expression analysis: Genesrping software (version 12.5; Agilent Technologies) was employed to finish the basic analysis. Differentially expressed genes were then identified through fold change. The threshold set for up- and down-regulated genes was a fold change>= 1.5 [2] Alternative splice analysis: Alternative splice analysis was conducted by Transcriptome Analysis Console (version1.0, Affymetrix). Differential exon or junction identified through splicing index. The threshold was splicing index >= 2.0 or < = -2.0 .
