6 protocols
Title: Affymetrix CEL analysis. Description:
RNA of the separated fluorescent various fractions (0-5% and 25-100% fluorescence fractions) was extracted using an RNAeasy RNA isolation kit (Qiagen) following the manufacturerﾒs instructions.
(Parameters: Extracted product = total_RNA, Amplification = none)
The experiment generated 2 sample groups: 0-5% and 25-100% fluorescence intensity cell fractions. The E coli strain used was a derivative of E. coli C600 (Appleyard, R. K. 1954. Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39, 440ﾖ452.) carrying a single chromosomal copy of miniTn5gusA-pgfp21 (Xi, C. W., Lambrecht, M., Vanderleyden, J. and Michiels, J. 1999. Bi-functional gfp-and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies. J. Microbiol. Meth. 35, 85ﾖ92.). This was introduced into E. coli C600 by electroporation with the suicide vector pFAJ1819 (Xi et al., 1999); colonies displaying GFP fluorescence were identified on agar plates under ultraviolet light (302 nm). A clone exhibiting stable GFP fluorescence after consecutive batch cultures was selected for further work, and was designated E. coli C600::mTn5gusA-pgfp21. Replicate cultures of E. coli C600::mTn5gusA-pgfp21 were inoculated at 2 hour intervals in nutrient broth at 37oC and monitored through to stationary phase by optical density measurements. One hour after reaching stationary phase, cells were harvested by centrifugation (10,000 x g 10 minutes) and pellets were washed three times in phosphate buffered saline (PBS, Sigma). These suspensions were transferred directly to a MoFlo flow cytometer and sorter (DakoCytomation) (70,000 sorts per second) and sorted for a 2 hour period. At the end of each 2 hour period the input suspension was changed for a fresh one. Each 2 hour sort experiment collected the 0-5% of fluorescence intensity cells for the majority of the time but at its mid-point was recalibrated to collect the 25-100% of fluorescence intensity cell fraction for approximately 20 minutes. Collections of sorted cells were made directly into tubes containing 5 mL of RNA Protect (Qiagen) until the tubes contained a total of 50 mL. Two replicate separation runs of 16 hours were conducted, and the collected fractions were pooled, lyophilised, and re-suspended in nuclease free water (Sigma).
Background correction,quantile normalization, and gene expression analysis were performed using RMA (Bolstad, B.M., Irizarry R. A., Astrand, M., and Speed, T.P. (2003)A Comparison of Normalization Methods for High Density Oligonucleotide Array Data Based on Bias and Variance. Bioinformatics 19(2):185-193).