|
| Status |
Public on Feb 08, 2017 |
| Title |
BloodAtTimepoint5,followingSleepInPhaseWithMelatoninForSubjectBB0008 |
| Sample type |
RNA |
| |
|
| Source name |
Subject_BB0008_FollowingSleep_InPhase_T5
|
| Organism |
Homo sapiens |
| Characteristics |
sampletype: Blood
sleep protocol: in phase
genotype: Per3_4/5
time sampletaken: 5
circadian phase: 192.64
|
| Treatment protocol |
2.5 ml of blood is drawn into the PAXGene tube at each sampling point. The tube is inverted 10 times to mix. The tube is then stored at room temperature for 4 hours in upright position. The tube is transferred to -50°C and placed lying down (never in upright position as this may cause the tube to crack) for storage at the university of Surrey Clinical Research Centre. The tube is transferred on ice to University of Surrey Faculty of Health and Medical Sciences for storage/processing
|
| Growth protocol |
Blood samples were collected via an indwelling venous cannula sited in the forearm for minimum discomfort and restriction of movement. Cannulae were kept patent by the use of sterile saline. Each sample tube was coded for subject and sample collection time (participant code/sample type/sequential number)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using a PAXgene Blood RNA Kit and a QiaCube robot (Qiagen, Hilden, Germany). The RNA was quantified and the A260/280 nm and A260/230 nm ratios were determined using a NanoDrop ND1000 spectrophotometer (Wilmington, DE). RNA quality was assessed using the Bioanalyzer 2100 (Agilent, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
cRNA was synthesized and fluorescently labelled with Cy3-CTP from 100 ng of total RNA using Agilent's Low Input Quick Amp Labeling Kit.
|
| |
|
| Hybridization protocol |
Labelled cRNA (1.65 μg) was hybridized on a Whole Human Genome 4 44K custom oligonucleotide microarray (G2514F, AMADID 026817; Agilent Technologies). Standard manufacturer's instructions for one-colour gene expression hybridization and washing steps were followed. The microarrays were hybridized at 65°C for 17 h in an Agilent hybridization oven with rotisserie at 10 rpm. The microarrays were washed with Agilent Wash Buffer 1, pre-warmed Wash Buffer 2 (37C) and acetonitrile according to the manufacturer’s instructions. The last washing step was performed with Agilent Stabilization and Drying Solution for 30 sec.
|
| Scan protocol |
The processed microarrays were scanned using an Agilent Microarray Scanner with a resolution of 5 μm, exploiting the extended dynamic range feature. The two images derived from each slide scanned at 10% and 100% PMT were imported into Agilent Feature Extraction software (Version 10.7.1.1) for image analysis.
|
| Description |
Blood taken at timepoint 5 ,following sleep in phase with melatonin, for Subject BB0008
|
| Data processing |
Individual samples were filtered based on AgilentQC metrics of reproducibility statistics, minimum detection level estimates and feature flags. Samples with a median coefficient of variation of less than 10% in spike ins or non-control replicated probes (NCRPs) were retained and quantile-normalised using the R Bioconductor package limma. NCRPs along with their corresponding flags were averaged. Probes with more than 5 flagged samples within a subject in more than half of the total number of subjects were excluded. In addition, for time-series analyses, series with 2 consecutive or more than 2 missing time points were excluded.
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| |
|
| Submission date |
Jun 01, 2016 |
| Last update date |
Feb 08, 2017 |
| Contact name |
Emma Laing |
| E-mail(s) |
e.laing@surrey.ac.uk
|
| Organization name |
University of Surrey
|
| Street address |
Stag Hill
|
| City |
Guildford |
| ZIP/Postal code |
GU22 7XH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL15331 |
| Series (1) |
| GSE82113 |
Mistimed sleep disrupts circadian regulation of the human blood transcriptome - Heterozygotes |
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