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| Status |
Public on Sep 01, 2014 |
| Title |
Real-time quantitative PCR analysis of mouse pre-implantation embryos |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by RT-PCR
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| Summary |
Adult female mice (C57BL/6) were super-ovulated by intraperitoneal hormone injections with the interval of 48 hours between injections. The hormones administered were PMSG followed by hCG (5IU/0.1ml, Sigma-Aldrich). Following the second hormonal treatment, the females were mated with males (1:1, C57BL/6). Zygotes, 2-cell and 4-cell and blastocysts were collected 26, 48 and 52 hours post hCG. Prior to cell collection, the zona pelucida was removed by Tyrode s acid solution. The 2- and 4-cell embryos were then immersed in Trypsin solution (Invitrogen) with RNase inhibitor (1 IU/ul, Clontech) and BSA (50 ug/ul) for the separation of the blastomeres. The separated blastomeres were snap frozen, and maintained at -80 C until processed. Twelve zygotes, 20 2-cell and 9 4-cell embryos were collected for single cell qPCR. In addition, we prepared with a pool of 10 zygotes and a pool of 50 zygotes as positive controls. The cells and pools were subjected to reverse transcription with SuperScript VILO cDNA Synthesis kit (Life Technologies) as recommended by the manufacturer. DELTAgene Assays were custom designed for 94 genes associated with embryonic development or regulation of gene expression, as well as 2 housekeeping genes. The cDNA from cells and pools were subjected to target specific amplification for 20 cycles. Pre-amplified template was diluted and subjected to primer specific amplification with SooFast EvaGreen Supermix (Biorad) on 96.96 array chip and BiomarkHD System (Fluidigm). Relative quantification of gene expression (log2 space) was obtained by subtracting the Ct values from the baseline value of 22.
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| Overall design |
12 zygotes, 10 2-cell, and 9 4-cell mouse (C57BL/6) embryos were collected and multi-cell embryos were separated into blastomeres
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| Contributor(s) |
Biase FH, Zhong S |
| Citation(s) |
25096407 |
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| Submission date |
Jul 29, 2014 |
| Last update date |
Dec 03, 2014 |
| Contact name |
Fernando H Biase |
| E-mail(s) |
fbiase@vt.edu
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| Phone |
540-231-9520
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| Organization name |
Virginia Tech
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| Department |
School of Animal Sciences
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| Lab |
Biase lab
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| Street address |
175 West Campus Drive
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| City |
Blacksburg |
| State/province |
VA |
| ZIP/Postal code |
24061 |
| Country |
USA |
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| Platforms (1) |
| GPL19012 |
Zhong Mouse pre-implantation development PCR assay panel (96 genes) |
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| Samples (90)
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| Relations |
| BioProject |
PRJNA257006 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE59892_Zhong_fluidigm_RT-PCR_raw_cts.txt.gz |
43.6 Kb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
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