|
| Status |
Public on Sep 01, 2011 |
| Title |
5521RA |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
5521RA
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Reflected Amnion
labor status: term no labor TNL
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Amnion tissues were liquid nitrogen-pulverized using a mortar and pestle, and total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA), and treated with DNase. The quality of the total RNA was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE).
|
| Label |
Hy3
|
| Label protocol |
The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. 0.3 µg total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
common reference
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: pooled reflected and placental amnions (pool of all samples in this study)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Amnion tissues were liquid nitrogen-pulverized using a mortar and pestle, and total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA), and treated with DNase. The quality of the total RNA was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE).
|
| Label |
Hy5
|
| Label protocol |
The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. 0.3 µg total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY™ LNA array version 11.0 (Exiqon, Denmark), which contains capture probes targeting all miRNAs for human, mouse or rat registered in the miRBASE version 13.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria).
|
| Scan protocol |
After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA).
|
| Description |
Placental amnion and reflected amnion were obtained from singleton placentas of patients at term with no labor (TNL; n=5) and in labor (TIL; n=5). Approximately a quadrant of placental amnion was sampled in each placenta by blunt dissection. Reflected amnion samples were taken from regions at least 2 cm apart from the rupture sites and the placental margin, also by blunt dissection.
|
| Data processing |
The quantified signals were background corrected (Normexp with offset value 0 – Ritchie et al., 2007, Bioinformatics (2007), Vol. 23 no. 20) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. These steps were performed using the limma package of Bioconductor using the R language (2.12.1). Normalized Log2 Hy3/Hy5 values were averaged over all spots corresponding to a unique miRNA. Only miRNAs with 2 or more spots having a foreground signal greater than background were considered present in a given sample. miRNAs not present in at least 4 samples were not considered for differential expression analysis.
|
| |
|
| Submission date |
Feb 22, 2011 |
| Last update date |
Sep 01, 2011 |
| Contact name |
Adi Tarca |
| E-mail(s) |
atarca@med.wayne.edu
|
| Phone |
3135775305
|
| Organization name |
Wayne State University
|
| Department |
Perinatology Research Branch (NIH/NICHD)
|
| Street address |
3990 John R
|
| City |
Detroit |
| State/province |
MI |
| ZIP/Postal code |
48188 |
| Country |
USA |
| |
|
| Platform ID |
GPL7723 |
| Series (1) |
| GSE27441 |
Amniotic microRNA profile of normal delivery at term |
|
