Dataset features


Application: Gene expression microarray analysis
Number of samples: 4
Release date: Sep 4 2006
Last update date: Aug 8 2013
Access: Public
Taxon: Synechocystis sp. PCC 6803
Dataset link Time course response of Synechocystis PCC 6803 to dehydration/desiccation

Experimental Protocol

Goal - Compare the responses of cyanobacterium Synechocystis sp. PCC 6803 to UV-irradiation and dehydration/desiccation stress. The data here relate to dehydration/desiccation stress. Origin of biological sample - The axenic strain Synechocystis sp. PCC 6803 was obtained from the American Type Culture Collection (ATCC 27184). Special attention was given to maintain all the Synechocystis cultures under the same conditions to minimize environmental variation. Brief description - Ultraviolet (UV) light and desiccation stresses often co-occur in natural environments, making their combined effects important selective factors within microbial populations. The effects of these stresses on the cyanobacterium Synechocystis sp. PCC 6803 was assessed through transcriptional analyses using differential display and microarray assays. Experimental factors and design - Axenic Synechocystis cultures were grown at 25°C in BG-11 medium, on a rotary shaker at 70 rpm, at a photon flux density of 200 µmol photons m-2 s-1. At the midpoint of the exponential phase of growth (~ 10^6 to 10^7 cells ml-1), 25-ml aliquots of axenic cultures were transferred to glass Petri dishes with covers and pre-adapted overnight. The covers were removed and cultures were subjected to a linear rate of evaporation of (55% relative humidity maintained in the incubator). Samples were taken at 25, 20, 15, 12, 7, 3 and 0.5 ml of evaporated cultures and completely dried cultures; the latter corresponding to a total of 15 h of dehydration. As controls, RNA was obtained from Synechocystis cells grown without dehydration. Cells were disrupted with glass beads and RNA was extracted with a buffer of acidified phenol (65°C), sodium acetate pH 5.1, EDTA and sodium dodecyl sulfate), followed by ethanol precipitation. The RNA (10 µg) was annealed with 20 µg Random RT primer and reverse transcription into cDNA was achieved with Superscript II reverse transcriptase enzyme for 2h, at 42°C. The assay contained dGTP, dCTP, dATP and dTTP, each a final concentration of 0.5 mM. After hydrolysis of RNA with 0.1M NaOH and 10 mM EDTA, then neutralization, cDNAs were purified using a Quiagen QIAquick PCR purification kit as modified in the Genisphere 3DNA Array 350RP protocol. Two experimental replicates of RNAs from time points 12 ml, and two experimental replicates from 0.5 ml, of dehydration, were used for hybridization to microarrays.