|
| Status |
Public on Mar 02, 2011 |
| Title |
normal placenta 5 and 7 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
normal placenta of sample 5
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: placenta
physiological state: normal
sample #: 5
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using Trizol reagent (Life Technologies, Rockville, MD).Total RNA was quantified by UV absorption at 260 nm, and RNA quality was examined by Agilent 2100 bioanalyzer (Agilent technologies, USA).
|
| Label |
Cy3
|
| Label protocol |
Each RNA sample was labeled by an indirect method using the 3DNA Array 50 Kit (Genisphere, USA). Briefly, 20 μg total RNA was used to perform reverse transcription reaction with SuperScript Ⅱ RNase H- reverse transcriptase and specific primers (Invitrogen life technologies, USA). Then all synthesized tagged cDNA targets were purified by Microcon YM-30 column (Millipore, USA).
|
| |
|
| Channel 2 |
| Source name |
normal placenta of sample 7
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: placenta
physiological state: normal
sample#: 7
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using Trizol reagent (Life Technologies, Rockville, MD).Total RNA was quantified by UV absorption at 260 nm, and RNA quality was examined by Agilent 2100 bioanalyzer (Agilent technologies, USA).
|
| Label |
Cy5
|
| Label protocol |
Each RNA sample was labeled by an indirect method using the 3DNA Array 50 Kit (Genisphere, USA). Briefly, 20 μg total RNA was used to perform reverse transcription reaction with SuperScript Ⅱ RNase H- reverse transcriptase and specific primers (Invitrogen life technologies, USA). Then all synthesized tagged cDNA targets were purified by Microcon YM-30 column (Millipore, USA).
|
| |
|
| |
| Hybridization protocol |
Two-step hybridizations were performed. During the first hybridization, cDNA products was pipetted onto the array. Then, the array was incubated in the hybridization chamber in a water bath at 65℃ for 16 to 20 hours for the probe/target hybridization. At the second hybridization, the 3DNA dendrimer was pipetted onto the array and the array was submerged in a 65℃ water bath for only 2 to 3 hours for capture primer – 3DNA dendrimer hybridization. After hybridization, array was washed with SSC and SDS mixed buffer suggested by manufacturer’s instruction of 3DNA array 50 kit.
|
| Scan protocol |
Microarrays were scanned by using GenePix 4100A (Axon, Foster City, CA) and images were taken at a resolution of 5μm. GenePix Pro 5.1 software was used to analyze image data.
|
| Description |
Placental specimens were obtained from the same region of placentas immediately after delivery. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (IRB#96-0630B). The tissues were snap frozen in liquid nitrogen and stored at -80°C.
|
| Data processing |
Spots were first selected by (1) excluding spots defined as flag or absent (2) keeping only channels with signal - background / SD of background greater than 2 (3) keeping spot diameter greater 75μm (4) eliminating spots with intensity CV greater than 100 in ether channel. The log ratio of the last spots were calculated and normalized by pin-wised LOWESS fit normalization.
|
| |
|
| Submission date |
Mar 02, 2011 |
| Last update date |
Mar 02, 2011 |
| Contact name |
wei-chung cheng |
| E-mail(s) |
d928503@oz.nthu.edu.tw
|
| Organization name |
NTHU
|
| Street address |
adqwerqr ew
|
| City |
hsinchu |
| ZIP/Postal code |
300 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL5355 |
| Series (1) |
| GSE27646 |
Intra- and Inter-Individual Variance of Gene Expression in Human Placenta |
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