developmental stage: 6-cell stage human embryo tissue: preimplantation embryo
Extracted molecule
total RNA
Extraction protocol
RNA from single oocytes and embryos was purified using RNA Clean XP bead suspension (Agencourt Bioscience, Beckman Coulter, Danvers, USA). Genomic DNA is bound by the beads but released in only a small percentage as long as it is not sheared (data not shown). Therefore, every effort was made to minimize shearing. RNA was eluted in 20 µl water. Different percentages of the RNA isolations were used for amplification, according to the expected RNA content of embryos. From MII, 2-cell, 4-cell, 6-cell and 8-10 cell embryos, the entire RNA preparation was used (20µl); from morulas, 6 µl were used; from blastocycsts 3 µl; from ESC, 4 µl. Library preparation were performed following the distributor’s (Sigma-Aldrich, St.Louis, USA) recommendations for WTA2. SYBR Green (Sigma-Aldrich, St.Louis, USA) was added to the amplification reaction, which was performed in a CFX Real-time instrument (Bio-Rad), to monitor yield. When the SYBR Green signal reached a plateau after 21 cycles, the reaction was stopped. Amplified cDNA was purified and quantified.
Label
biotin
Label protocol
10 µg cDNA was subsequently fragmented by DNAseI and biotinylated by terminal transferase (Affymetrix, Santa Clara, USA).
Hybridization protocol
Hybridization, washing, staining and scanning of Affymetrix Human Gene ST 1.0 arrays were performed following the manufacturer’s recommendations.
Scan protocol
Scanned images (DAT files) were transformed into intensities (CEL files) by GCOS (Affymetrix, Santa Clara, USA).
Description
All embryos were frozen at the pronuclear stage, 18 to 20 h after insemination. After thawing, embryos were allowed to develop until the required stage in a sequential culture system (G1/G2, Vitrolife AB, Goteborg, Sweden) system, at 37°C and 6% CO2. Embryos were monitored every 12 h to detect developmental progression. Once the embryos reached the appropriate stage of development, their zona pellucida (ZP) was removed by exposure to pronase (5mg/ml; Roche, Basel, Switzerland) at 37°C. Dezoned oocytes and embryos were immediately plunged in 45 µl of Lysis Buffer (20 mM DTT, 10 mM TrisHCl, 0.5% SDS, 1 mg/ml Proteinase K) preheated at 65°C. The tube was maintained at 65°C for 15 minutes and placed at -80°C for later processing.
Data processing
Statistical analysis of the data was performed using the affy package in R (Gautier et al., 2004). Briefly, raw CEL files (which will be submitted to the GEO database upon manuscript acceptance) were imported with gene annotation from NetAffix and data was normalized with GCRMA and summarized at the gene level (32,321 genes). We performed gene expression profiles in 2 batches, and corrected for batch effect using the ComBat algorithm in R (Johnson et al., 2007).
