lymphoblastoid cell line from R, one out of seven of sensitive group, 50*10e6 cells
Biomaterial provider
established in department of Otolaryngology / Head-Neck Surgery, VUmc, Amsterdam, the Netherlands
Treatment protocol
bleomycin 4hr 5 mU/ml
Extracted molecule
total RNA
Label
Cy3
Label protocol
Non-amplified total RNA (75 µg) was used for preparation of labelled cDNA essentially as described (Buermans HP, et al. Physiol Genomics 2005;21:314-23). In brief, anchored oligo-dT [(dT)20-VN] primer was used and SuperScript II Reverse Transcriptase (Invitrogen), with incorporation of amino-allyl dUTP(Ambion, Huntingdon, UK). RNA was hydrolysed with 0.1 mM NaOH (70°C, 10 min) and after neutralizing with 0.1 mM HCl the samples were diluted in water and transferred onto a Microcon YM-30 filter (Millipore, Billerica, MA, USA) to purify and concentrate the cDNA. Before labelling with the dye, the dry cDNA pellet was dissolved in 9 ml of NaHCO3/NaCO3 (50 mM, pH 9.0) and incubated at room temperature for 1 hr in the dark with 2 ml aliquots Cy 3(Fluorolink Cy 3 Monofunctional Dye (Amersham). Unbound dye was removed using the Qiaquick PCR purification kit (Qiagen, Venlo, the Netherlands)according to the manufacturer’s protocol.
Pooled RNA from a number of lymphoblastoid cell lines (with and without bleo treatment) and head and neck cancer cell lines.
Biomaterial provider
established in department of Otolaryngology / Head-Neck Surgery, VUmc, Amsterdam, the Netherlands
Treatment protocol
untreated
Extracted molecule
total RNA
Label
Cy5
Label protocol
see label protocol of ch1 (Cy5 instead of Cy3)
Hybridization protocol
According to Buermans HP, et al. Physiol Genomics 2005;21:314-23
Scan protocol
According to Buermans HP, et al. Physiol Genomics 2005;21:314-23
Description
Not applicable
Data processing
ImaGene feature extraction (Biodiscovery Ltd, Marina del Rey, CA, USA) was used to record spot intensities. The signal mean was taken to represent the actual spot intensity, after subtraction of the mean background values. All spot were log2 transformed and intensities lower than 10, typical for the empty spots, were classified as missing. Next for each micro-array, the spot intensities were Z score normalized (Cheadle C, et al. J Mol Diagn 2003;5:73-81), while missing values (some 6% overall) were handled so as to minimize their effect on statistics. Specifically, such values were set equal to the mean of the micro-array distribution after Z score normalization, thus equal to zero. So for each micro-array, Z scores were computed for each green labeled spot intensity according to(2log(G) - mu) / sigma, where mu is the mean and sigma the standard deviation of all 2log(G) of the micro-array. Likewise the Z score was computed for each red labeled spot intensity and the result subtract from the ‘green’ Z score for each spot on the micro-array. These differences were then again Z score normalized to obtain the so called Z ratio for each spot.This information will soon be available in a publication.
The signal mean of each channel was taken to represent the actual spot intensity, after subtraction of the mean background values. Intensities lower than 10 were neglected and all values above 10 were log2 transformed .The spot intensities were Z score normalized (Cheadle C, et al. J Mol Diagn 2003;5:73-81), while missing values (some 6% overall) were handled so as to minimize their effect on statistics. Specifically, such values were set equal to the mean of the micro-array distribution after Z score normalization, thus equal to zero. So for each micro-array, Z scores were computed for each ch1 spot intensity according to(2log(G) - mu) / sigma, where mu is the mean and sigma the standard deviation of all 2log(G) of the micro-array. Likewise the Z score was computed for each ch2 labeled spot intensity and the result subtracted from the green Z score for each spot. These differences were then again Z score normalized to obtain the so called Z ratio for each spot.This Z-ratio defines VALUE.
