Surehyb chamber

Samples for gene expression analysis were labeled using Agilent Quick-Amp labeling Kit one-color. Aliquots of 500 ng of each sample were incubated with a reverse transcription mixture at 40 °C and converted to double stranded cDNA primed by oligo (dT) with a T7 polymerase promoter. Synthesized double stranded cDNA was used as template for cRNA generation. The cRNA was generated by in vitro transcription, and the dye Cy3 CTP (Agilent) was incorporated during this step, both carried out at 40 °C. Labeled cRNA was cleaned up, and its quality was assessed using NanoDrop® ND-1000 UV-Vis spectrophotometer. The specific activity determination was based on the cRNA concentration and dye incorporation.
The labeled cRNA samples were hybridized onto an AZ-specific microarray chip, AMADID: 043310^{34 (link)} designed by Genotypic Technology Pvt. Ltd (Bangalore, India). Aliquots of 1650 ng of Cy3-labeled samples were fragmented and hybridized by using the Gene Expression Hybridization kit of Agilent. Hybridization was carried out in an Agilent SureHyb chamber at 65 °C for 16 h. The hybridized slides were washed using Agilent Gene Expression wash buffers, and scanned using the Agilent Scanner, Part Number G2600D. Data extraction from images was performed by using Feature Extraction software of Agilent V-11.5.

mRNA from MCF-7 cells transfected with pcDNA3.1-PinX1 and empty vector was purified from the total RNA using a mRNA-ONLY Eukaryotic mRNA Isolation Kit (Epicentre, USA); subsequently, Cyanine-3-CTP (NEB, USA) was incorporated and the fluorescent cRNA of each sample was linearly amplified and transcribed using a Quick Amp Labeling Kit, One-Color (Agilent, USA). The labeled cRNA was then purified using a RNeasy Mini Kit (Qiagen, Germany). The hybridization solution for each sample was prepared using a Gene Expression Hybridization Kit (Agilent, USA) and 1.5 μg of the labeled cRNA. The hybridization solution was applied on the Human LncRNA Array V2.0 (Arraystar, USA) in a SureHyb chamber (Agilent, USA), and the hybridization was conducted in a hybridization oven (Agilent, USA) at 65°C for 17 hours.

Gene expression analysis was performed with an Agilent® SurePrint G3 Human GE 8x60K Microarray (Agilent Technologies, AMADID 39494) with the following dual-color design. The test samples were labeled with Cy5, whereas the control samples were labeled with Cy3 using a two-color Agilent labeling kit (Low Input Quick Amp Labeling Kit 5190-2306) adapted for a small amount of total RNA (100 ng total RNA per reaction). Hybridization was then performed on the microarray using 825 ng of each linearly amplified cRNA-labeled Cy3 or Cy5 sample following the manufacturer’s protocol (Agilent SureHyb Chamber; 1650 ng of labeled extract; duration of hybridization: 17 h; 40 µL per array; temperature: 65 °C). After washing in acetonitrile, slides were scanned using an Agilent G2565 C DNA microarray scanner under default parameters (100° PMT, 3 µm resolution, 20 °C in a free ozone concentration environment). Microarray images were analyzed by using Feature Extraction software version (10.7.3.1) from Agilent Technologies. Default settings were used.