|
| Status |
Public on Sep 25, 2014 |
| Title |
Cord_blood_Espoo_10 |
| Sample type |
RNA |
| |
|
| Source name |
Cord blood
|
| Organism |
Homo sapiens |
| Characteristics |
country: Finland
sample month: January
gender: Male
hla risk category (np=neutral/protective, m=medium, h=high, vh=very high): H
dr3-dq2: -
dr4-dq8: +
gestational week: 42
hybridization batch: 1
|
| Growth protocol |
Umbilical cord blood was drawn into Tempus Blood RNA tubes according to the manufacturer´s protocol and then stored in −70 °C until analyzed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total whole-blood RNA was extracted from the samples using Tempus Spin RNA isolation kit (Applied Biosystems) according to manufacturer's instructions. RNA quality and quantity was determined using NanoDrop ND-2000 (Thermo Scientific) and Experion Automated Electrophoresis System (Bio-Rad Laboratories).
|
| Label |
biotin
|
| Label protocol |
50 ng of total RNA was processed to cDNA with Ovation RNA amplification system v2, including the Ovation whole blood reagent (NuGEN Technologies). The amplified cDNA was subsequently biotin-labelled and fragmented with Encore biotin module (NuGEN Technologies).
|
| |
|
| Hybridization protocol |
Samples were hybridized to GeneChip Human Genome U219 array plate (Affymetrix) with manufacturer´s protocols for using the GeneTitan Hybridization, Wash and Stain Kit for 3’ IVT Array Plates (Affymetrix). GeneTitan MC Instrument (Affymetrix) was used to hybridize, wash, stain, and scan the arrays.
|
| Scan protocol |
GeneTitan MC Instrument was used to scan the arrays. The probe cell intensity data was summarized with Affymetrix GeneChip Command Consol 3.1.
|
| Data processing |
The intensity data was pre-processed using robust multi-array averaging (RMA). The absent call filtering was done by determining a threshold value empirically for each sample by fitting a two-component GMM for all non-control probesets. A probeset was filtered out if it was absent in at least 50 % of the samples from Espoo, Tartu and Petrozavodsk. Batch correction was done by applying the ComBat analysis method. Additional 15 rehybridized samples (in a separate data set) were utilized in this correction but excluded from further analysis steps. These intensities were then used to detect differential expression by fitting a linear model and computing a moderated t-statistic for each present probeset for all three contrasts: Espoo vs. Petrozavodsk, Tartu vs. Petrozavodsk and Espoo vs. Tartu. Gender, pregnancy week, month of birth and HLA risk class were included as confounding factors in the model.
|
| |
|
| Submission date |
Dec 18, 2013 |
| Last update date |
Sep 26, 2014 |
| Contact name |
Essi Laajala |
| E-mail(s) |
essi.laajala@btk.fi
|
| Organization name |
Turku Centre for Biotechnology
|
| Street address |
Tykistökatu 6
|
| City |
Turku |
| ZIP/Postal code |
20520 |
| Country |
Finland |
| |
|
| Platform ID |
GPL13667 |
| Series (2) |
| GSE53471 |
Standard of hygiene and immune adaptation in newborn infants [113 cord blood RNA samples] |
| GSE53473 |
Standard of hygiene and immune adaptation in newborn infants |
|
