|
Status |
Public on Sep 01, 2015 |
Title |
Control Embyro and LIF-siRNA-treated embryo Replicate 1 |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
The LIF-siRNA-treated embryo
|
Organism |
Mus musculus |
Characteristics |
strain: B6CBF1 age: 4 d tissue: whole embryo
|
Treatment protocol |
The embryos were either injected with 1pl of 1.0 fmole siRNA into the cytoplasma. The embryos injected with scrambled siRNA (1pl of 1.0 fmole) act as a positive control. The embryos were then incubated in an atmosphere of 5% CO2 at 37℃ and monitored daily using an optical microscope.
|
Growth protocol |
Zygotes were collected from the oviducts of female mice successfully mated in the laboratory and placed into wells with fresh human tubal fluid medium. Embryos in the two-pronucleus (2PN) stage were obtained by incubating the zygotes in an atmosphere with 5% CO2 at 37℃ for 4 h.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
Label |
Cy5
|
Label protocol |
Total RNA (1.5–3 μg) of LIF-siRNA injected embryo and scrambled-siRNA injected embryo were reverse transcribed with Superscript II RNase H- Reverse Transcriptase (Gibco BRL, Grand Island, NY, USA) to generate Cy5 (red) and Cy3 (green) fluorescent dye-labeled complementary DNA (cDNA) probes, respectively.
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|
|
Channel 2 |
Source name |
The scrambled siRNA-treated embryo
|
Organism |
Mus musculus |
Characteristics |
strain: B6CBF1 age: 4 d tissue: whole embryo
|
Treatment protocol |
The embryos were either injected with 1pl of 1.0 fmole siRNA into the cytoplasma. The embryos injected with scrambled siRNA (1pl of 1.0 fmole) act as a positive control. The embryos were then incubated in an atmosphere of 5% CO2 at 37℃ and monitored daily using an optical microscope.
|
Growth protocol |
Zygotes were collected from the oviducts of female mice successfully mated in the laboratory and placed into wells with fresh human tubal fluid medium. Embryos in the two-pronucleus (2PN) stage were obtained by incubating the zygotes in an atmosphere with 5% CO2 at 37℃ for 4 h.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
Label |
Cy3
|
Label protocol |
Total RNA (1.5–3 μg) of LIF-siRNA injected embryo and scrambled-siRNA injected embryo were reverse transcribed with Superscript II RNase H- Reverse Transcriptase (Gibco BRL, Grand Island, NY, USA) to generate Cy5 (red) and Cy3 (green) fluorescent dye-labeled complementary DNA (cDNA) probes, respectively.
|
|
|
|
Hybridization protocol |
The labeled probes were hybridized to Agilent Mouse Oligo Microarrays, a commercial cDNA microarray (Agilent Technologies, Taipei, Taiwan) containing 9652 immobilized cDNA fragments.
|
Scan protocol |
Fluorescence intensities of Cy3 and Cy5 targets were measured and scanned separately using a Dig Luminescent Detection Kit (Roche, Taipei, Taiwan) and Fluor-S MAZ Multi-Imager System (Bio-Rad, Hercules, CA).
|
Description |
Biological replicate 1 of 4. Control embryonic stem cells, untreated, harvested after several passages.
|
Data processing |
Data analysis was performed using Agilent Feature extraction software Version A.6.1.1 (Agilent Technologies). The normalized log ratio =LOG(SQRT( mean background light intensity of Cy3 channel*mean background light intensity of Cy5 channel),2). The criteria for determination of differentially expressed genes were that the absolute value of the ratio of Cy5 to Cy3 was greater than two-fold (up-regulated) or less than 0.5-fold (down-regulated), and the signal value of fluorescence intensity of either Cy3 or Cy5 was greater than 1,000 intensity.
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|
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Submission date |
Aug 05, 2015 |
Last update date |
Sep 01, 2015 |
Contact name |
Jer-Yuh Liu |
E-mail(s) |
jyl@mail.cmu.edu.tw
|
Phone |
+886-912610015
|
Organization name |
China Medical University
|
Department |
Graduate Institute of Cancer Biology
|
Street address |
Hsueh-Shih Road
|
City |
Taichung |
ZIP/Postal code |
40402 |
Country |
Taiwan |
|
|
Platform ID |
GPL872 |
Series (1) |
GSE71736 |
The requirement of leukemia inhibitory factor or epidermal growth factor for pre-implantation embryogenesis |
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