|
| Status |
Public on Oct 30, 2010 |
| Title |
blastomere from 5-cell embryo E4.M1 |
| Sample type |
RNA |
| |
|
| Source name |
Blastomere 1. Embryo 4
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: blastomere
developmental stage: 5-cell embryo
|
| Treatment protocol |
Single blastomeres from 5- to 8- cell human embryos, and inner cell mass and trophectoderm from blastocysts were biopsied and transferred to lysis buffer for later cDNA amplification protocol
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The biopsied single blastomeres, inner cell masses and trophectoderms were collected in cell lysis buffer for RNA denaturalization. First-strand cDNAs were synthesized by retro-transcription with the SSIII (Invitrogen) and were then tailed with poly(dA) by TdT (Invitrogen, CA, USA). The cDNA products were divided and the second strand synthesized with another oligo(dT)-tagged primers and amplified. PCR products were purified with the DNA Clean & Concentrator™ Kit (Zymo Research, CA, USA),and subjected to another PCR amplification reaction containing the primers bearing the T7 promoter. PCR products were then purified with the DNA Clean & Concentrator™ Kit (Zymo Research, CA, USA) and electrophoresed and gel purified with the ZymoClean™ Gel DNA Recovery Kit (Zymo Research, CA, USA). Purified products were divided and subjected to a final PCR cycle and purified with the DNA Clean & Concentrator™ Kit (Zymo Research, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
300 ng of cDNA were used to produce Cyanin 3-CTP-labeled cRNA using the Quick Amp Labelling Kit, One-Color (Agilent p/n 5190-0442). "One-Color Microarray-Based Gene Expression Analysis" protocol Version 5.7 (Agilent p/n G4140-90040) was followedfor 16 hours in the IVT reaction and the resulting cRNA purified. Yield and Cy3 specific activity were measured by spectrometry (Nanodrop, DW, USA) and cRNA length evaluated by the mRNA 6000 Nano Bioanalyzer assay.
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| |
|
| Hybridization protocol |
Up to 3000 ng of Cy3 labeled cRNA were hybridised for 20 hours to the Whole Human Genome Oligo Microarray Kit.
|
| Scan protocol |
Arrays were scanned in an Agilent microarray Scanner (Agilent G2565BA) according to the manufacturer´s protocol.
|
| Description |
Amplified cDNA from single blastomere
Global gene expression of single blastomere from 5-cell embryo
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software 9.5.3, grid template 014850_D_F_20080627, following the Agilent Protocol GE1-v5_95_Feb07.Samples were first filterd by comparison to the negative sample microarray and those considered not to have signal were excluded from teh analysis. Agilent Processed Signals were standardized across arrays usign quantile normalization.
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| |
|
| Submission date |
May 27, 2010 |
| Last update date |
May 28, 2010 |
| Contact name |
Amparo Galan |
| E-mail(s) |
agalan@cipf.es
|
| Organization name |
Prince Felipe Research Centre (CIPF)
|
| Street address |
Avda Autopista del Saler 16, 3
|
| City |
Valencia |
| ZIP/Postal code |
46021 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE22032 |
Defining Cell Fate And Embryonic Genome Activation By Global Single-Cell cDNA Analysis of Blastomeres From 5-to 8-Cell Human Embryos. |
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