NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM362060 Query DataSets for GSM362060
Status Public on Dec 31, 2009
Title PBMC_Fasted_Con_14
Sample type RNA
 
Channel 1
Source name PBMC, Fasted, Control, 14
Organism Rattus norvegicus
Characteristics Wistar, Gender: Male, Age: 6 months, Tissue: PBMC
Treatment protocol Immediately after blood collection, PBMC were isolated by Ficoll gradient separation.
Growth protocol Blood samples (1.5-2.5 mL) were collected from rats in feeding conditions from the safena vein, using heparine in NaCl (0.9%) as anticoagulant.
Extracted molecule total RNA
Extraction protocol Total RNA from PBMC samples was extracted using Tripure Reagent (Roche Diagnostic Barcelona, Spain) and purified with a Quiagen RNesay Mini Kit spin columns (Izasa SA, Barcelona, Spain).
Label Cy5
Label protocol 0.5 μg RNA of each sample was reverse transcribed using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent), according to the manufacturer’s protocol (all materials and reagents are from Agilent Technologies, Palo Alto, CA, USA unless stated otherwise). Half of the cDNA sample (10 µL) was used for linear RNA amplification and labelling with Cy5 or Cy3. All reactions were performed using half of the amounts indicated by the manufacturer. Briefly, a transcription master mix was prepared (7.65 µL nuclease-free water; 10 µL 4x transcription buffer; 3 µL 0.1 M DTT; 4 µL NTP mix, 3.2 µL 50% PEG; 0.25 µL RNaseOUT; 0.3 µL inorganic pyrophosphatase; 0.4 µl T7 RNA Polymerase; 1.2 µL cyanine 3-CTP or cyanine 5-CTP, total volume 30 µL). 30 µL of transcription mastermix was added to10 µL cDNA. In vitro transcription and labelling were carried out at 40°C for 2 h. The labelled cRNA samples were purified using Qiagen Rneasy mini-spin columns (Qiagen, Venlo, The Netherlands). Dye incorporation and cRNA concentration was measured using the ‘micro-array measurement mode’ of the Nanodrop spectrophotometer (NanoDrop Technologies Wilmintog, Delaware, USA). Yield of each individual sample was >825 ng and specific activity >8.0 pmol Cy3 or Cy5 per µg cRNA. All Cy3 cRNAs were pooled to serve as a standard reference pool.
 
Channel 2
Source name Reference RNA. This consists of a pool of equal molar total RNA samples of all groups analyzed.
Organism Rattus norvegicus
Characteristics Wistar, Gender: Male, Age: 6 months, Tissue: PBMC
Extracted molecule total RNA
Extraction protocol Total RNA from PBMC samples was extracted using Tripure Reagent (Roche Diagnostic Barcelona, Spain) and purified with a Quiagen RNesay Mini Kit spin columns (Izasa SA, Barcelona, Spain).
Label Cy3
Label protocol 0.5 μg RNA of each sample was reverse transcribed using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent), according to the manufacturer’s protocol_ch2 (all materials and reagents are from Agilent Technologies, Palo Alto, CA, USA unless stated otherwise). Half of the cDNA sample (10 µL) was used for linear RNA amplification and labelling with Cy5 or Cy3. All reactions were performed using half of the amounts indicated by the manufacturer. Briefly, a transcription master mix was prepared (7.65 µL nuclease-free water; 10 µL 4x transcription buffer; 3 µL 0.1 M DTT; 4 µL NTP mix, 3.2 µL 50% PEG; 0.25 µL RNaseOUT; 0.3 µL inorganic pyrophosphatase; 0.4 µl T7 RNA Polymerase; 1.2 µL cyanine 3-CTP or cyanine 5-CTP, total volume 30 µL). 30 µL of transcription mastermix was added to10 µL cDNA. In vitro transcription and labelling were carried out at 40°C for 2 h. The labelled cRNA samples were purified using Qiagen Rneasy mini-spin columns (Qiagen, Venlo, The Netherlands). Dye incorporation and cRNA concentration was measured using the ‘micro-array measurement mode’ of the Nanodrop spectrophotometer (NanoDrop Technologies Wilmintog, Delaware, USA). Yield of each individual sample was >825 ng and specific activity >8.0 pmol Cy3 or Cy5 per µg cRNA. All Cy3 cRNAs were pooled to serve as a standard reference pool.
 
 
Hybridization protocol Hybridization was performed by preparing a 2x cRNA target solution containing 825 ng Cy5-labeled cRNA, 825 ng Cy3-labeled pool cRNA and 11 µL 10x blocking agent in a total volume of 52.8 µL. Then 2.2 µL fragmentation buffer was added and incubated at 60°C for 30 min. Fragmentation was stopped by adding 55 µL 2x GEx hybridization buffer HI-RPM and hybridized on 4x 44K G4131F whole genome Agilent arrays (Agilent Technologies, Inc. Santa Clara, CA) for 17 h at 65°C in Agilent hybridization chambers in an Agilent hybridization oven rotating at 10 rpm. After hybridization the arrays were subsequently washed with ‘GE wash buffer 1’ for 1 min at room temperature, ‘GE wash buffer 2’ for 1 min at approximately 37°C, acetonitrile for 1 min at room temperature and 30 s at room temperature with ‘stabilization and drying solution’ according to manufacturers protocol (Agilent Technologies).
Scan protocol Arrays were scanned with an Agilent Microarray Scanner (Agilent Technologies). Spot intensities were quantified using Feature extraction 8.5 (Agilent Technologies).
Description The data table contains normalized sample (Cy5) signal intensity, and not normalized log ratio (test/reference) data
Data processing Median density values and background values of each spot were extracted for both the experimental samples (Cy5) and the reference samples (Cy3). Data was exported into GeneMaths XT (Applied Maths, Sint-Martens-Latem, Belgium) for analysis. Spots with an average Cy5 signal intensity, over all arrays, lower than 2-fold average Cy5 local background were discarded. Subsequently, the remaining Cy5 signal intensities were normalized against the Cy3 reference using RIKILT normalization protocol (Pellis L, et al. (2003) Physiol Genomics 16: 99-106).
 
Submission date Jan 21, 2009
Last update date Jan 22, 2009
Contact name Jaap Keijer
E-mail(s) jaap.keijer@wur.nl
Phone +31-317-484136
Fax +31-317-484077
Organization name Wageningen University
Department Human and Animal Physiology
Street address Marijkeweg 40
City Wageningen
State/province gld
ZIP/Postal code 6708PG
Country Netherlands
 
Platform ID GPL4135
Series (1)
GSE14497 Diet and feeding condition induced gene expression in rat peripheral blood mononuclear cells

Data table header descriptions
ID_REF
VALUE Normalized sample (Cy5) signal intensity for genes with signal above 2 times background level (blanks or spots below background threshold, are discarded before normalisation).

Data table
ID_REF VALUE
1
2
3
4
5
6
7
8
9
10
11
12
13
14 11.900636
15
16 9.504592
17 16.814381
18
19 7.032719
20 9.475628

Total number of rows: 45220

Table truncated, full table size 472 Kbytes.




Supplementary file Size Download File type/resource
GSM362060.txt.gz 14.0 Mb (ftp)(http) TXT
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap