The strain A. ferrooxidans ATCC 23270 was cultured in 9K basal medium (initial pH 2.0) added with FeSO4•7H2O (44.7 g/L) or S0 (10 g/L) as energy substrate. The 9K basal medium contained the following components: (NH4)2SO4, 3.0 g/L; MgSO4•7H2O, 0.5 g/L; K2HPO4, 0.5 g/L; KCl, 0.1 g/L, Ca(NO3)2, 0.01 g/L. The inoculum density of A. ferrooxidans was 5.6×105 cells/mL. It was cultivated aerobically at 30 °C in a rotary platform at 170 rpm.
Growth protocol
Control group was set up without fluoride. Experimental group were treated by adding 476 mM sodium fluoride solution to the basal medium, with the final fluoride concentration 4.8 mM, when cells grown to the mid-log phase (40 hours) .
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, USA) and RNeasy mini kit (Qiagen, Valencia, USA). RNA quality was assessed by Bioanalyzer (Agilent, USA) and spectrophotometer (Nanodrop Technologies).
Label
Cy3
Label protocol
The pure RNA was converted to cDNA with random primers following the manufacturer’s protocol for the ImProm-II™ Reverse Transcription System (Promega Corporation, Madison, USA), and purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany).The cDNA of samples with/without fluoride treatment were labeled using Cy3-/Cy5-dUTP fluorescent dyes
Channel 2
Source name
Continuous cultured control samples at 10 min without fluoride introduced
The strain A. ferrooxidans ATCC 23270 was cultured in 9K basal medium (initial pH 2.0) added with FeSO4•7H2O (44.7 g/L) or S0 (10 g/L) as energy substrate. The 9K basal medium contained the following components: (NH4)2SO4, 3.0 g/L; MgSO4•7H2O, 0.5 g/L; K2HPO4, 0.5 g/L; KCl, 0.1 g/L, Ca(NO3)2, 0.01 g/L. The inoculum density of A. ferrooxidans was 5.6×105 cells/mL. It was cultivated aerobically at 30 °C in a rotary platform at 170 rpm.
Growth protocol
Control group was set up without fluoride. Experimental group were treated by adding 476 mM sodium fluoride solution to the basal medium, with the final fluoride concentration 4.8 mM, when cells grown to the mid-log phase (40 hours) .
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, USA) and RNeasy mini kit (Qiagen, Valencia, USA). RNA quality was assessed by Bioanalyzer (Agilent, USA) and spectrophotometer (Nanodrop Technologies).
Label
Cy5
Label protocol
The pure RNA was converted to cDNA with random primers following the manufacturer’s protocol for the ImProm-II™ Reverse Transcription System (Promega Corporation, Madison, USA), and purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany).The cDNA of samples with/without fluoride treatment were labeled using Cy3-/Cy5-dUTP fluorescent dyes
Arrays scanned by a GenePix Personal 4100A scanner (AXON instruments, Inc, USA) and then was converted to digital signals by Genepix Pro 6.1 software.
Description
RNA from control samples were compared to the experimental samples taken at 10, 30, 60, 120 and 240 min after fluoride added.
Data processing
A grid of individual circles defining the location of each DNA spot on the array was superimposed on the image to designate each fluorescent spot to be quantified. The mean signal intensity was determined for each spot. The local background signal was subtracted automatically from the hybridization signal of each separate spot also computed for each spot to discriminate true signals from noise. The SNR ratio was also calculated based on the following formula: SNR=(signal intensity – background)/standard deviation of background, in which the background measurement refers to the local spot background intensity and the standard deviation of background was calculated across all pixels measured by the ImaGene software. The SNRs from four replicate data sets were then averaged to represent the SNR for a particular probe. A commonly accepted criterion for the minimum signal (threshold) that can be accurately quantified is SNR > 2. Spots that appeared to be lower than the threshold value were removed from the data set.
