6 protocols
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])
10 ug of total RNA, with the addition of spike-in controls (ArrayControl RNA Spike, Ambion) was reversely transcribed using anchored-oligo(dT)20, aminoallyl-modified dNTPs and SuperScript III reverse transciptase from SuperScript Plus Indirect cDNA Labeling System (Invitrogen). Aminoallyl-modified cDNA from the first step was precipitated with 2,5 vol. of 96% ethanol and 1/10 vol. of 3M Sodium Acetate, pH 5.2. Amino-modified cDNAs from all patients and healthy volunteer samples were labeled with AlexaFluor 647 fluorescent dye (Invitrogen). The common reference sample (RNA extracted from HL60 cells, obtained from Poznan University of Life Sciences) was labeled with AlexaFluor 555 (Invitrogen). Labeled cDNAs were on-column purified (MinElute Reaction Cleanup Kit, Qiagen).
(Parameters: Amount of nucleic acid labeled = 10, Amplification = none, Mass unit = Micro gram)
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 40?C, 5 min. (II) 2x SSC at RT, 5 min., (III) 0.2x SSC at RT, 5 min. Then the slides were dried through centrifugation.
(Parameters: Chamber type = OTHER: PerkinElmer_HybArray12 hyb station , Quantity of label target used = 1, Mass unit = Micro gram, time = 15, Tiny time unit = hours, Volume = 120, Volume unit = Micro litre, temperature = 50)
Peripheral blood and bone marrow samples were collected into EDTA-containing tubes in the Department of Hematology, Poznan Universityof Medical Sciences, Poznan, with patient consent. Mononuclear cells were separated in density gradient centrifugation (Gradisol L, Aqua-Medica, Poland) and washed twice with 1xPBS (Phosphate Buffered Saline, Ca and Mg-free,BIOMED, Poland). The cell pellet was suspended in Lysis Buffer from mirVanamiRNA Isolation Kit (Ambion) and frozen in -80 degrees C.
Total RNA was then extracted according to mirVana miRNA Isolation Kit manual (Ambion) and digested with DNase (TURBO DNA-free kit, Ambion). The quality of RNA was verified using Bioanalyzer 2100 (Agilent).
(Parameters: Extracted product = total_RNA, Amplification = none)
The HL-60 cell line (human promyelocytic leukemia cells) stock was obtained from Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw. HL60 cells were cultured in the Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences. The cells were maintained in RPMI 1640 medium supplemented with 2mM glutamine, 20% Heat-Inactivated Foetal Bovine Serum and 50ug/l gentamicin, sulfate by replacement of medium and with subsequent resuspension at 1 X 10(5) viable cells/ml when culture reached 1 X 10(6) cells/ml. For RNA isolation the cells were washed with PBS, suspended in Lysis Buffer from mirVana miRNA Isolation Kit (Ambion) and frozen in -80 degrees C.