|
| Status |
Public on Jul 20, 2011 |
| Title |
passive smoker 215, cord blood |
| Sample type |
RNA |
| |
|
| Source name |
umbilical cord blood
|
| Organism |
Homo sapiens |
| Characteristics |
smoking status: passive smoker
age (years): 34.4969199178645
maternal bmi: 23.7953599048186
parity: 2
gestational age (weeks): 38
mode of delivery: vaginal
placental weight (g): 620
placental volume (cm3): 1500
newborn weight (g): 2270
newborn length (cm): 43
apgar score (5s): 10
baby's sex: M
maternal blood cotinin (ng/ml): 0.46
cord blood cotinin (ng/ml): no sample
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Umbilical cord bloods were sampled and processed using LeukoLOC Total RNA Isolation System (Ambion, Austin, TX, USA) according to the manufacturer manual. The system employs filter-based leukocyte-depletion technology to isolate leukocytes from whole blood and RNAlater™ (Ambion) to stabilize the cells on the filter. By removal of red blood cells, the RNA purified from captured leukocytes is inherently depleted of globin mRNA, which improves sample quality for expression profiling and other applications. The middle parts of placenta sections were frozen in RNA Later solution (Ambion) and stored at –20°C until RNA isolation. Placental total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer instructions. Integrity of the total RNAs was assayed by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and only samples with RNA integrity number (RIN) above 7.0 were used for gene expression profiling.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA was prepared from 200 ng of total RNA using Illumina TotalPrep RNA Amplification Kit (Ambion). In vitro transcription (IVT) was performed at 37°C for 14 hours.
|
| |
|
| Hybridization protocol |
750 ng of biotinylated cRNA was hybridized to the array for 17 hours according to the manufacturer manual. Final staining was performed with streptavidin conjugated to Cy-3 fluorescent dye.
|
| Scan protocol |
Arrays were scaned using BeadArray Reader (Illumina) and bead level data were extracted by BeadStudio Software (Illumina).
|
| Description |
5-cm away from the site of cord insertion
|
| Data processing |
Bead summary data were imported into R statistical environment (www.r-project.org) and normalized by quantile method in Lumi package. Only probes, which reached detection P-value < 0.01 in all samples, were used for further analyses. Differentially expressed genes were analysed in the Limma package. A linear model was fitted for each gene given a series of arrays using lmFit function. Using Benjamini and Hochberg method P-values were corrected for multiple testing.
|
| |
|
| Submission date |
Jun 16, 2011 |
| Last update date |
Jul 20, 2011 |
| Contact name |
Hana Bruchova |
| E-mail(s) |
Hana.Bruchova@uhkt.cz
|
| Phone |
+420221977306
|
| Fax |
+420221977371
|
| Organization name |
Institute of Hematology and Transfusion
|
| Department |
Molecular Genetics
|
| Lab |
Genomics
|
| Street address |
U nemocnice 1
|
| City |
Prague 2 |
| ZIP/Postal code |
128 20 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL6883 |
| Series (1) |
| GSE30032 |
Deregulation of Gene Expression induced by Environmental Tobacco Smoke Exposure in Pregnancy |
|
