normalization data transformation protocol
mAdb Data Processing Protocol (v. 2). Calculation Method: After background correction, ratios were median centered based on signals greater than 100 in both channels and linear transformed to obtain the log and linear values given in the data table. ID_REF = VALUE = log2 ratio (Cy5/Cy3) with values flagged as Error removed and left blank
array scanning protocol
Creator: GenePix Pro 184.108.40.206, Scanner: GenePix 4000B , ScanPower: 100;; 100, LaserPower: 2.88;; 3.7, Temperature: 30.97
For pre-hybrization, apply 40 ul of pre-hybridization buffer (5X SSC, 0.1% SDS, 1% BSA) to the array and incubate at 42°C for at least 30 minutes and up to an hour. Wash off the pre-hybridization solution by rapidly plunging the slide in distilled water for 2 minutes, then transfer slide to 100% isopropanol for 2 minutes. Allow slide to air dry completely prior to use. (Can spin dry if in a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying before setting up hybridization.) For hybridization, combine Cy3 and Cy5 labeled targets together (~11 ul recovered for each). Add 1 µl herring sperm DNA (8-10 ug/ul) and 0.2 ul tNRA (8-10 ug/ul). Denature target at 100°C for 1 minute, then snap cool on ice. (Final volume should be about 12 ul.) Make fresh 2X Formamide hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) and warm to 42°C just before adding to samples. Add 12 ul of 2X F-hyb buffer to samples. Load 25 ul sample onto microarray. Add 20 ul of 3X SSC to wells in hyb chamber to maintain humidity. Incubate overnight (12-16 hours) at 42°C in water bath or hybridization oven. After hybridization of slides, wash slides for 2 minute in 2X SSC with 0.1% SDS (on rocking platform), for 2 minute in 1X SSC (with rocking), for 2 minutes in 0.2X SSC (on rocking platform), for 1 minute in 0.05X SSC, and spin for 3 minutes at 650 rpm to dry.
Cy5: 4ug total RNA is converted to cDNA using Invitrogen random primers and superscript III at 48C in presence of 10-fold excess of Cy5-dCTP above unlabeled dCTP according to manufacturer's instructions. Cy3: 4ug total RNA is converted to cDNA using Invitrogen random primers and superscript III at 48C in presence of 10-fold excess of Cy3-dCTP above unlabeled dCTP according to manufacturer's instructions.
nucleic acid extraction protocol
Extracted molecule total RNA. Extraction protocol Trizol MTb Extraction. Extraction method: Trizol-QIAgen RNeasy Kit. Mycobacterium tuberculosis was rapidly harvested and cell pellet suspended in Trizol, subjected to 2-3 rounds of bead beating (0.1mm zirconia beads), phase separation performed with CHCl3 and RNA purified with the Quiagen RNeasy clean up kit using Qiagen on-column DNase to remove contaminating DNA.