|
| Status |
Public on Apr 27, 2007 |
| Title |
Day 25 extra-embryonic membrane and endometrium (BPL_25A2) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Day25 endometrium
|
| Organism |
Bos taurus |
| Characteristics |
Bovine endometrium of day 25 of gestation
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated from the samples using ISOGEN (NipponGene) according to the manufacturer's instructions. PolyA RNA was isolated from the samples using Oligotex-dT30 Super (Roche) according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
Two-microgram of pooled polyA+ RNA of tissue obtained from pregnant cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Bioscience) by using Superscript II reverse transcriptase (Invitrogen) to yield Cy5 fluorescence-labeled cDNA probe. After a 2-h reverse transcription reaction at 42C, RNA was degraded by addition of 1.5 micro-l of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 micro-l 1N HCl.The Cy5 cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 micro-g Salmon sperm DNA, 20 micro-g polyA and 20 micro-g yeast RNA in a Microcon YM-30 column (Millipore). The concentrated probe mix was adjusted with TE to a volume of 10 micro-l, denatured at 100C for 2 min, spun for a few min and made to a final hybridization volume of 15 micro-l with 2.55 micro-l 20XSSC and 0.45 micro-l 10% SDS and kept at room temperature until used.
|
| |
|
| Channel 2 |
| Source name |
Day25 extra-embryonic membrane
|
| Organism |
Bos taurus |
| Characteristics |
Bovine extra-embryonic membrane of day 25 of gestation
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated from the samples using ISOGEN (NipponGene) according to the manufacturer's instructions. PolyA RNA was isolated from the samples using Oligotex-dT30 Super (Roche) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Two-microgram of pooled polyA+ RNA of tissue obtained from pregnant cows was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Bioscience) by using Superscript II reverse transcriptase (Invitrogen) to yield Cy3 fluorescence-labeled cDNA probe. After a 2-h reverse transcription reaction at 42C, RNA was degraded by addition of 1.5 micro-l of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 micro-l 1N HCl.The Cy3 cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 micro-g Salmon sperm DNA, 20 micro-g polyA and 20 micro-g yeast RNA in a Microcon YM-30 column (Millipore). The concentrated probe mix was adjusted with TE to a volume of 10 micro-l, denatured at 100C for 2 min, spun for a few min and made to a final hybridization volume of 15 micro-l with 2.55 micro-l 20XSSC and 0.45 micro-l 10% SDS and kept at room temperature until used.
|
| |
|
| |
| Hybridization protocol |
Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (Wako) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the Cy3 & Cy5, were then applied to the microarray, placed a cover slip carefully and incubated at 65C for 14 - 16 h in a custom slide chamber. After incubation for 14 - 16 h at 65C, the slides were washed in 2 x SSC / 0.01% SDS for 2 min, followed by washing in 0.2 x SSC/0.01% SDS and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed.
|
| Scan protocol |
Hybridization images were immediately scanned by a GenePix 4000B laser scanner (Axon Instrument) and analyzed with GenePix Pro 4.0 computer software.
|
| Description |
This data is utilized for gene expression analysis of bovine placenta.
|
| Data processing |
subtracted from spot intensity data. The subtracted intensity data were subjected to global normalization.
|
| |
|
| Submission date |
Feb 21, 2007 |
| Last update date |
Apr 27, 2007 |
| Contact name |
Koichi Ushizawa |
| E-mail(s) |
ushizawa@affrc.go.jp
|
| Phone |
+81-29-838-8633
|
| Fax |
+81-29-838-8606
|
| Organization name |
National Institute of Agrobiological Sciences
|
| Department |
Animal Science Department
|
| Lab |
Reproductive Biology Unit
|
| Street address |
Ikenodai 2
|
| City |
Tsukuba City |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-8602 |
| Country |
Japan |
| |
|
| Platform ID |
GPL1221 |
| Series (1) |
| GSE7096 |
cDNA microarray data of bovine placenta |
|
| Data table header descriptions |
| ID_REF |
|
| CY5_RAW |
Cyanine5 raw signal intensity median |
| CY5_SD |
Cyanine5 raw signal intensity standard deviation |
| CY5_BKD_RAW |
Cyanine5 background signal intensity median |
| CY5_BKD_SD |
Cyanine5 background signal intensity standard deviation |
| CY3_RAW |
Cyanine3 raw signal intensity median |
| CY3_SD |
Cyanine3 raw signal intensity standard deviation |
| CY3_BKD_RAW |
Cyanine3 background signal intensity median |
| CY3_BKD_SD |
Cyanine3 background signal intensity standard deviation |
| FLAGS |
Measure of a feature (hybridized spot) quality |
| PRE_VALUE |
Unlogged signal measurement for each feature (hybridized spot) |
| VALUE |
Logged Cy5/Cy3 signal measurement for each feature (hybridized spot) |
