Gender: female, Age: 51 years, ACR score at 14 weeks after first infliximab injection: 70%
Extracted molecule
total RNA
Extraction protocol
The blood (2.5 ml x 2 tubes) was drawn using PAXgene tubes (QIAGEN, Hilden, Germany). The total RNA extraction and purification were carried out according to the manufacturer’s protocol.
Label
Cy5
Label protocol
Then total RNA (1 mg) from patients was transcribed and amplified to produce amplified sample RNA (aRNA) using the MessageAmp aRNA kit according to the manufacturer’s instructions. Next, an external control RNA mixture (LD, GP, and RL) was added to both the sample and reference aRNA (9 mg each). These mixed sample and reference aRNA were labeled using SuperScript II kit (Invitrogen, Carlsbad, CA) with random hexamer (TaKaRa, Kyoto, Japan) with Cy3-dUTP and Cy5-dUTP (PerkinElmer, Boston, MA), respectively.
The blood (2.5 ml x 2 tubes) was drawn using PAXgene tubes (QIAGEN, Hilden, Germany). The total RNA extraction and purification were carried out according to the manufacturer’s protocol.
Label
Cy3
Label protocol
Then total RNA (1 mg) from patients was transcribed and amplified to produce amplified sample RNA (aRNA) using the MessageAmp aRNA kit according to the manufacturer’s instructions. Next, an external control RNA mixture (LD, GP, and RL) was added to both the sample and reference aRNA (9 mg each). These mixed sample and reference aRNA were labeled using SuperScript II kit (Invitrogen, Carlsbad, CA) with random hexamer (TaKaRa, Kyoto, Japan) with Cy3-dUTP and Cy5-dUTP (PerkinElmer, Boston, MA), respectively.
Hybridization protocol
Competitive hybridization of Cy3-labeled reference and Cy5-labeled sample cDNA on the microarray was carried out according to Brown and Yanofsky[10] with chamber systems (Agilent Technologies, Palo Alto, CA).
Scan protocol
Slides were scanned five times with five different power ranges using ScanArray 5000 (PerkinElmer, Boston, MA).
Description
no additional information
Data processing
The data was converted from tif image data to signal using ImaGene software (BioDiscovery, El Segundo, CA) for further statistical analysis. Five file data of each four spot data corresponding each gene were merged to establish the single representative data for each gene (Patent pending: PCT/JP03/06677).
mRNA expression profile in peripheral blood cells of RA patients following treatment with an anti-TNFa mAb, infliximab
Data table header descriptions
ID_REF
VALUE
The Cy5 (patient sample)/Cy3 (reference sample) ratio of each mRNA signal was calculated for further analysis. The global Lowess normalization method was also applied before ratio calculation.
