After selection of Oxa-resistant cell lines, the analysis of resistance degree and cross resistance to other drugs was assessed by using MTT colorimetric assay. All cell lines were grown in P60 dishes and total RNA from each sample isolated by using the RNeasy Mini kit (Qiagen) and were exhaustively treated with RNase-free DNAse I. RNA quantification were performed using NanoDrop spectophotometer (Nucliber).
Extracted molecule
total RNA
Extraction protocol
All cell lines were grown in P60 dishes and total RNA from each sample isolated by using the RNeasy Mini kit (Qiagen) and were exhaustively treated with RNase-free DNAse I. RNA quantification were performed using NanoDrop spectophotometer (Nucliber).
Label
Cy5,Cy3
Label protocol
Labeling of total RNA was performed using 3DNA Array-350 Detection kit according to manufacturer's protocol (Genisphere, Hatfield, PA). Total RNA was reverse transcribed using reverse transcription (RT) primers. Briefly, 5 µg of total RNA and 1 µl of Cy3 or Cy5 capture sequence were brought to 11 µl with RNase free water and incubated for 10 min at 80°C. In a separate microtube 4 µl of 5x RT buffer, 1 µl dNTP mix, 2 µl 0.1M DTT, 1 l of Superase In TM Rnase inhibitor and 1µl of Superscript II reverse transcriptase were combined and the reaction was incubated 2 h at 42°C. The reaction was terminated by adding 3.5 µl of 0.5 M NaOH/50 mM EDTA and incubating it at 65°C for 10 min. The reaction was neutralized with 5 µl 1 M Tris-Cl, pH 7.5. The Cy3 and Cy5 reactions were then combined and precipitated with 15 µg of linear acrylamide, 250 µl 3 M ammonium acetate, and 875 µl 100% ethanol at -20 °C for 1 hour. Following precipitation the pellet was dried with speed-vac. In preparation for hybridization the cDNA pellet was resuspended in 29 µl RNase free water deionized distilled water. Then, 29 µl hybridization buffer (vial 12) were added to the mix and incubated at 80 °C for 10 min, followed by 55 °C for 45 min. This is the Final Hybridization Mix for loading on the array.
After selection of Oxa-resistant cell lines, the analysis of resistance degree and cross resistance to other drugs was assessed by using MTT colorimetric assay. All cell lines were grown in P60 dishes and total RNA from each sample isolated by using the RNeasy Mini kit (Qiagen) and were exhaustively treated with RNase-free DNAse I. RNA quantification were performed using NanoDrop spectophotometer (Nucliber).
Extracted molecule
total RNA
Extraction protocol
All cell lines were grown in P60 dishes and total RNA from each sample isolated by using the RNeasy Mini kit (Qiagen) and were exhaustively treated with RNase-free DNAse I. RNA quantification were performed using NanoDrop spectophotometer (Nucliber).
Label
Cy3,Cy5
Label protocol
Labeling of total RNA was performed using 3DNA Array-350 Detection kit according to manufacturer's protocol (Genisphere, Hatfield, PA). Total RNA was reverse transcribed using reverse transcription (RT) primers. Briefly, 5 µg of total RNA and 1 µl of Cy3 or Cy5 capture sequence were brought to 11 µl with RNase free water and incubated for 10 min at 80°C. In a separate microtube 4 µl of 5x RT buffer, 1 µl dNTP mix, 2 µl 0.1M DTT, 1 l of Superase In TM Rnase inhibitor and 1µl of Superscript II reverse transcriptase were combined and the reaction was incubated 2 h at 42°C. The reaction was terminated by adding 3.5 µl of 0.5 M NaOH/50 mM EDTA and incubating it at 65°C for 10 min. The reaction was neutralized with 5 µl 1 M Tris-Cl, pH 7.5. The Cy3 and Cy5 reactions were then combined and precipitated with 15 µg of linear acrylamide, 250 µl 3 M ammonium acetate, and 875 µl 100% ethanol at -20 °C for 1 hour. Following precipitation the pellet was dried with speed-vac. In preparation for hybridization the cDNA pellet was resuspended in 29 µl RNase free water deionized distilled water. Then, 29 µl hybridization buffer (vial 12) were added to the mix and incubated at 80 °C for 10 min, followed by 55 °C for 45 min. This is the Final Hybridization Mix for loading on the array.
Hybridization protocol
The microarrays used in our study were the Human 19K Oligo Array from Center for Applied Genomics (University of Medicine of New Jersey). Microarray slides were prehybridized in buffer containing 5x SSC, 25% formamide and 0.1% SDS at 42 °C for 45min and washed by dipping in isopropanol. The slides were then dipped in distilled water and dried with compressed air. The Final Hybridization Mix was added to the prehybridized slide. A 22 x 60 mm coverslip was cleaned with compressed air and then gradually placed on the slide to form a thin layer of labeled targets. To maintain humidity inside the chamber, 20 l 2x SSC was added to the two reservoir wells. The chamber was then tightly sealed and incubated at 55 °C water bath overnight for 16-20 h. Slide was then removed from the chamber, washed for 10 min sequentially at 42 ºC in 2x SSC/0.2% SDS buffer, at room temperature in 2x SSC buffer and at room temperature in 0.2x SSC buffer, rinsed in distilled water for 5 s, and dried by compressed air. Then, we prepare the 3DNA Array 350 Capture Reagent (Vial 1) by thawing the vial in the dark at room temperature for 20 minutes, vortexing at the maximum setting for 3 seconds and microfuge briefly and incubating at 50-55°C for 10 minutes. Simultaneously we resuspended the 2X Hybridization Buffer (Vial 6) by heating to 70°C for at least 10 minutes. For each array, we prepare a 3DNA Hybridization Mix containing 2.5ul of 3DNA Array 350 Capture Reagent-1 (Vial 1), 2.5ul of 3DNA Array 350 Capture Reagent-2 (Vial 1) , 25ul of 2X Hybridization Buffer (Vial 6) and 20 ul of water. Gently vortex and briefly microfuge the 3DNA Hybridization Mix. We incubate the 3DNA Hybridization Mix first at 75-80°C for 10 minutes, and then we add the 3DNA Hybridization Mix to a prewarmed microarray, taking care to leave behind any precipitate at the bottom of the tube. To maintain humidity inside the chamber, 20 l 2x SSC was added to the two reservoir wells. The chamber was then tightly sealed and incubated at 60 °C water bath for 4 h. Slide was then removed from the chamber, washed for 10 min sequentially at 65 ºC in 2x SSC/0.2% SDS buffer, at room temperature in 2x SSC buffer and at room temperature in 0.2x SSC buffer, rinsed in distilled water for 5 s, and dried by compressed air.
Scan protocol
All slides were scanned immediately with an Affymetrix GMS 418 Array Scanner using appropriate gains on the photomultiplier tube to obtain the highest intensity without saturation. A 16 bit TIFF image was generated for each channel, Cy3 and Cy5. The image analysis was performed with Imagene 4.1 software (BioDiscovery) to quantify the signal intensities. Data from spots not recognized by the Imagene analysis software were excluded from further considerations (empty, poor and negative spots). Data from the spots visually identified as flawed (<15% in all arrays) were also removed. Only the spots with flag value equal to 0 were included in the analysis.
Description
Each cell line was analyzed in duplicate, with RNA reference (Stratagene) as reference sample and labeled each biological condition once by Cy3 and once by Cy5. Taking the average of two arrays thus labeled, cancel the dye effect on any particular gene. In total we used 16 slides.
Data processing
The data sets were imported into Microsoft Excel in order to analyze the quality for each feature. Two parameters were used to asses the data filtration: difference between median and mean under 20% for each feature and intensity feature median greater than background mean plus 2 standard deviation. Normalization data was performed with ArrayNorm 1.7.2 software by using Lowess and Dye Swap Sim Fix Filter methods. Visualization of results was performed with the normalized data using average linkage and Euclidean distance as a measurement of similarity using GENESIS 1.5.0 Software (Alexander Sturn, Institute for Genomics and Bioinformatics, Graz University of Technology). ANOVA was used as statistical function to determine gene expression differences between resistant (cluster R) and sensitive (cluster S) cell groups. The differences were considered statistically significant when two-sided p-values were less than 0.01.
