5 protocols
Slides are placed in an Axon 4200AL scanner connected to the GenePix Pro 6.0 image acquisition and analysis software package. Slides are scanned at 635nm and 532nm wavelength (respectively for Cy5 and Cy3). An initial pre-scan of the slide is performed to determine success of hybridisation and to allow the user to define the boundaries of the array features. PMT gain settings are adjusted at this step to ensure that the overall red-to-green ratio is similar. The slides are scanned and the resulting images are saved as a .TIF file for analysis and feature extraction using GenePix Pro. (Parameters: Scanning hardware = OTHER: Axon 4200 AL, Scanning software = GenePix Pro [Axon Instruments])
Cy3 and Cy5 targets quantity were normalized at 150 pmol, mixed and thereafter concentrated using Microcon YM-30 (Millipore). Samples volumes were adjusted to 20ul in nuclease-free water, and used for Agilent hybridization protocol( Two color microarray-based gene expression analysis protocol session Hybridization) (Parameters: Chamber type = OTHER: Agilent technology chambers, Quantity of label target used = 300, Mass unit = Nano gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 65)
RNA samples (5 ug) were indirectly labelled using Superscript indirect cDNA kit (InVitrogen) with a mixture of random hexamer (pdN6) according to the suppliers instructions. Target qualities and concentrations were determined by spectroscopy at 260nm, 280nm and 650nm (Parameters: Amplification = none, Mass unit = Micro gram)
Bacterial cell pellet, resuspended in 400 ul buffer R (glucose 10% ; 12.5 mM Tris ph 7.6; EDTA 10 mM), 60 ul EDTA 0.5M and 500 ul phenol ph 4.5 (Interchim) is broken in the presence of 0.4g glass beads (200-300 ums; Sigma) by two bursts (Speed:6.0; Time 30s) in Fast Prep. The sample is then centrifuged and the supernatant successively extracted by: 1) 1 ml Trizol, 100 ul chloroform/isoamylic alcohol 24/1 (v/v); 2) 200 ul chloroform/isoamylic alcohol 24/1. The final supernatant is pelleted using isopropanol; the pellet is rinsed by Ethanol 70%, dried and dissolved in 50 ul RNase free water. RNA samples are controlled for concentration with Nanodrop and for quality with Agilent Bioanalyzer 2100 and RNA 6000 nanokit. (Parameters: Extracted product = total_RNA, Amplification = none)
THmedium. Temperature 30C. DO= 0.3-0.4 (mid-exponential). (Parameters: time unit = seconds, min temperature = 30, temperature unit = C, media = TH)