5 protocols
Hybridized slides were scanned on a GenePix 4100A scanner (Axon Instruments, Inc.). Images were acquired at 10-um resolution, and the background-subtracted median spot intensities were determined using GenePix Pro 5.1 image analysis software (Axon Instruments, Inc.). Spots with anomalies were discarded and excluded from further analysis.
(Parameters: Scanning hardware = GenePix Personal 4100A [Axon Instruments], Scanning software = GenePix 4100A [Axon Instruments])
Pseudomonas putida DOT-T1E cells were grown overnight on M9 minimal medium with glucose as a carbon source and used to inoculate fresh medium with and without 100ug/ml ampicillin. All cultures grew exponentially and when a turbidity of 0.6 at 660 nm was reached, cells were harvested and immediately processed for RNA extraction.
(Parameters: start time = 6, time unit = hours, min temperature = 30, temperature unit = C, media = M9 (glucose))
Total RNA was extracted by using TRI Reagent (Ambion, ref. 9738) as recommended by the manufacturer except that Tripure Isolation reagent was preheated at 70 degrees C followed by purification with RNeasy columns (QIAGEN, cat no. 74104). RNA concentration was determined spectrophotometrically and its integrity was assessed by agarose gel eletrophoresis.
(Parameters: Extracted product = total_RNA, Amplification = OTHER: Reverse transcriptase)
25 ug of total RNA were primed with 3 ug of pd(N)6 random hexamers (Amersham, Cat. No. 27-2166-01). cDNA synthesis was performed at 42 degrees C for 2 h in a 30-ul reaction volume containing 0.5 mM (each) dATP, dCTP, and dGTP; 0.25 mM (each) dTTP and aminoallyl-dUTP (aa-dUTP; Sigma Cat. No. A0410); 10 mM DTT; 40 U of RNaseOUT (Invitrogen, ref. 10777-019); and 400 U of SuperScript II reverse transcriptase (RT) (Invitrogen, ref. 18064-014) in RT reaction buffer. The reaction was stopped by adding 10 ul of 50 mM EDTA and the RNA template was hydrolyzed with the addition of 10 ul of 1 N NaOH followed by incubation at 65 degrees C for 15 min. Samples were then neutralized by adding 25 ul of 1M HEPES (pH 7.5) and the hydrolyzed RNA and residual dNTPs were removed using QIAquick PCR purification columns (QIAGEN, ref. 28104) according to the manufacturer's recommendations, except that the Tris-based elution buffer (EB) supplied with the kit was replaced with phosphate elution buffer (4 mM KPO4 pH 8.5) to avoid interferences with the subsequent labeling. cDNA samples were dried in a Speed-Vac to completion. Dried aminoallyl-labeled cDNA was resuspended in 9 ul of 0.1 M sodium carbonate buffer (pH 9.0), mixed with Cy5 (samples of cells grown with chloramphenicol) or Cy3 (samples of cells grown without stressor) fluorescent dye (mono-reactive NHS-ester; Amersham Biosciences, Cat No. PA25001), and allowed to couple for 2 h at room temperature in the dark. After coupling, the reaction was quenched with 4.5 ul of 4 M hydroxylamine for 15 min. Finally labeled cDNA probes were again purified with QIAquick PCR purification columns. Labeling efficiency was assessed using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies).
(Parameters: Amount of nucleic acid labeled = 25, Amplification = none, Mass unit = Micro gram)
Prior to the hybridization process, the microarray slides were blocked by immersion into 5 x SSC (1x SSC is 0.15 M NaCl; 15 mM sodium citrate, pH 7), 0.1% (wt/vol) SDS, 1% (wt/vol) bovine serum albumin for 1 h at 42 degrees C. Then, the slides were washed by two successive immersions in MilliQ water at room temperature, followed by a final wash with isopropanol. The slides were spin-dried by centrifugation at 1,500 x g for 5 min, and used within the next hour. Equal amounts of Cy3- and Cy5-labeled cDNAs, one of them corresponding to the control and the other one to the problem to be analyzed, were mixed, dried in a Speed-Vac and reconstituted in 35 ul of hybridization buffer (5x SSC, 25% [vol/vol] formamide, 0.5% [wt/vol] SDS, 5x Denhardt’s solution, 5% [wt/vol] dextransulfate) preheated to 42ºC. The labelled probe was denatured at 98 degrees C for 3 min, applied onto the microarray slide and covered with a glass lid. The slide was then introduced in a humidified hybridization chamber (AHC ArrayIt Hybridization Cassette; Telechem International, Inc.) and incubated for 18 to 20 h in a water bath at 42 degrees C, preserved from light. Following hybridization, the microarrays were washed by gentle agitation in 2x SSC, 0.1% [wt/vol] SDS at 42 degrees C for 5 min, followed by a 5-min wash at room temperature in 1x SSC, two 5-min washes in 0.2x SSC, and a final 5-min wash in 0.1x SSC. Finally, the slides were spin-dried in a centrifuge at 1,500 x g for 5 min, and scanned.
(Parameters: Chamber type = OTHER: AHC ArrayIt Hybridization Cassette; Telechem International, Inc, Quantity of label target used = 6, Mass unit = Micro gram, time = 20, Tiny time unit = hours, Volume = 35, Volume unit = Micro litre, temperature = 42)