Blood samples obtained from patients were suspended in RNAlater Stabilization Reagent (Qiagen) and centrifuged (10 min, 3000 x g). Cell pellet was resuspended in buffer containing 1% beta-mercaptoethanol and homogenized by pulling through 0.9 mm syringe needle.
The slides were scanned with the ScanArrayExpress (Perkin Elmer) at 5-?m resolution and two lasers, corresponding to AlexaFluor 555 and AlexaFluor 647 excitation ranges. Raw data files were generated in GenePixPro6.1 (Molecular Devices).
(Parameters: Scanning hardware = ScanArray [PerkinElmer], Scanning software = ScanArray Express [PerkinElmer])
Hybridization was performed in automatic hybridization station HybArray12 (Perkin Elmer) using step-down hybridization protocol (5h in 50°C, 5h in 45°C and 5h in 40°C). Three subsequent wash steps were applied in the machine as follows: (I) 2x SSC, 0.1 % SDS at 35°C, 5 min. (II) 2x SSC at RT, 10 min., (III) 0.2x SSC at RT, 10 min. Then the slides were dried through centrifugation.
(Parameters: Chamber type = OTHER: PerkinElmer-HYbArray12 hyb station, Quantity of label target used = 300, Mass unit = Nano gram, time = 15, Tiny time unit = hours, Volume = 100, Volume unit = Micro litre, temperature = 50)
300 ng of RNA was transcribed into cDNA using SuperScriptII reverse transcriptase (Invitrogen), oligo(dt) and SMARTII primers. 8 spike-in controls (Ambion) were added into RT reaction in the following concentrations: 1, 5, 10, 20, 40, 60, 100, 500 pg/ul. Purified via ethanol precipitation cDNA was PCR-amplified using Pwo SuperYield DNA polymerase (Roche). PCR product served as a template for synthesis of antisense ssDNA incorporated with aminoallyl-modified nucleotides. Amino-allyl-modified ssDNA was labeled with the dye-pair from SSTM Plus Indirect cDNA Labeling System (Invitrogen): ssDNAs from patient samples were labeled with AlexaFluor 647 while ssDNAs from control samples with AlexaFluor 555 and on-column purified (MinElute Reaction Cleanup Kit, Qiagen).
(Parameters: Amount of nucleic acid labeled = 300, Amplification = PCR, Mass unit = Nano gram)
RNA was extracted from homogenized cells using AllPrep DNA/RNA Mini Kit (Qiagen), DNase I (Qiagen) digested and precipitated in -80ºC in 2.5 volume of 98% ethanol and 3 M NH4OAc, pH 4.8. The quality of RNA was verified using Bioanalyzer 2100 (Agilent).
(Parameters: Extracted product = total_RNA, Amplification = none)