Mark Fellmann, Div. Of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany
Treatment protocol
chemically synthesized siRNAs (Qiagen, Hilden, Germany) were transfected with HiPerFect (Qiagen, Hilden, Germany) according to standard transfection procedure
Growth protocol
cells were cultivated in Minimum Essential Media (1x) (MEM, Invitrogen, Grand Island, NY, USA), supplemented with 10 % fetal calf serum (FCS), 50 U/ml penicillin, and 50 µg/ml streptomycin sulfate, 1% non-essential amino acids (Invitrogen, Grand Island, NY, USA) and 100 µg/ml insulin bovine
Extracted molecule
total RNA
Extraction protocol
total RNA was extracted using Rneasy mini-prep kit (Qiagen, Hilden, Germany; 2µg total RNA was amplified one round based on T7 RNA Polymerase (Agilent low RNA Input Amplification Kit)
Label
Cy3
Label protocol
direct fluorescent labelling of 2 µg amplified RNA, anneling of 0.5µg Random Primer and reverse-transcribed into cDNA with Superscript III reverse transcriptase for 1h at 42°C in the presence of 0.4 mM dGTP, 0.4 mM dATP, 0.4 mM dTTP, 0.24 mM dCTP, 0.125 mM Cy3-dCTP. After hydrolysis of RNA in 0.2 M NaOH, Cy3-labeled probes were purified with Microcon YM-30 column.
Mark Fellmann, Div. Of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany
Treatment protocol
chemically synthesized siRNAs (Qiagen, Hilden, Germany) were transfected with HiPerFect (Qiagen, Hilden, Germany) according to standard transfection procedure
Growth protocol
cells were cultivated in Minimum Essential Media (1x) (MEM, Invitrogen, Grand Island, NY, USA), supplemented with 10 % fetal calf serum (FCS), 50 U/ml penicillin, and 50 µg/ml streptomycin sulfate, 1% non-essential amino acids (Invitrogen, Grand Island, NY, USA) and 100 µg/ml insulin bovine
Extracted molecule
total RNA
Extraction protocol
total RNA was extracted using Rneasy mini-prep kit (Qiagen, Hilden, Germany; 2µg total RNA was amplified one round based on T7 RNA Polymerase (Agilent low RNA Input Amplification Kit)
Label
Cy5
Label protocol
direct fluorescent labelling of 2 µg amplified RNA, anneling of 0.5µg Random Primer and reverse-transcribed into cDNA with Superscript III reverse transcriptase for 1h at 42°C in the presence of 0.4 mM dGTP, 0.4 mM dATP, 0.4 mM dTTP, 0.24 mM dCTP, 0.125 mM Cy5-dCTP. After hydrolysis of RNA in 0.2 M NaOH, Cy5-labeled probes were purified with Microcon YM-30 column.
Hybridization protocol
ch1 and ch2 together are solved in 50 µl 1x DIG-Easy hybridization buffer (Roche Diagnostics, Mannheim, Germany), containing 10x Denhardts solution and 2 ng/µl Cot1-DNA (Invitrogen); sample was denaturated at 65°C for 2min, hybridzation of arrays were performed in Corning chambers 14h at 37°C; slides were washed twice in 1xSSC+0.1SDS and once in 0.1xSSC+ 0.1SDS before air pressure drying and scanning.
Scan protocol
arrays were scanned with the GenePix 4000B microarray scanner and analyzed using GenePix Pro 4.1 software (Axon Instruments)
Description
siRNA_AKT2_DKFZ_Replicate2
Data processing
raw data processing was performed using ArrayMagic (Buness et al., 2005) including VSN normalization method; after removal of bad quality clones, generalized log ratios from 26618 cDNA clones were given in the data table
