origin: monitoring well from TCE contaminated site site: B sampling campaign: 5 well: P3 treatment: after ERD (enhanced reductive dechlorination) sample type: environmental sample
Growth protocol
Groundwater samples were collected by SITA Remediation Company (Lyon, France) from a contaminated site (site B) by direct pumping from three monitoring wells (P1, P2 and P3) drilled in the deep aquifer. P1: well located upstream to the contamination source. P2: well in the contamination source. P3 : well located downstream to the contamination source.
Extracted molecule
total RNA
Extraction protocol
Groundwater samples were transferred to the laboratory, filtered under vacuum and collected onto a 47 mm 0.2 µm pore size GTTP filter (Millipore). Filters were then cut in pieces and placed into 900 µL of nucleic extraction buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl pH 8.0) as described by Vetriani (Vetriani, C., Tran, H.V. and Kerkhof, L.J. (2003) Fingerprinting Microbial Assemblages from the Oxic/Anoxic Chemocline of the Black Sea. Appl. Environ. Microbiol., 69, 6481-6488). In this protocol, freeze-thaw cycles were d by beat betting 1 min at 30 Hz with 1 g of glass beads of ≤106 μm in diameter (Sigma-Aldrich). Total RNA were extracted using a standard phenol-chloroform-isoamyl alcohol (25:24:1) method followed by a second extraction with an equal volume of chloroform. Nucleic acids were then precipitated, washed in 70% ethanol and resuspended in nuclease-free water. Co-extracted DNA was removed by digestion with 4U of (RNAse-free) DNase I (DNA-free, Ambion) at 37°C for 35 min.
4 µg of total RNA mixture were subjected to an mRNA enrichment protocol using the MicrobExpress kit (Ambion Applied Biosystems), which employs capture hybridization by magnetic separation to remove 16S and 23S rRNA. In a second step, antisense amino-allyl dUTP marked RNA (aRNA) was obtained by amplification with both the MessageAmp II-Bacteria and the Amino-Allyl MessageAmp II kits (Ambion).
Label
Cy3
Label protocol
Antisense mRNA obtained from groundwater samples were labelled with Cy3 fluorescent dye (CyDyeTM Post-Labeling Reactive Dye Packs, GE Healthcare) following the manufacturer's instructions.
Hybridization protocol
For each hybridization experiment, labeled antisenses RNA (Cy3) were vacuum dried and resuspended in the NimbleGen Hybridization Buffer. The arrays were hybridized on a 4-bay NimbleGen Hybridization System (Roche NimbleGen) at 42°C for 72 hr. Arrays were washed with NimbleGen wash buffers I, II and III according to vendor protocols.
Scan protocol
Arrays were scanned using a Scanner Innoscan 900AL (Innopsys, Carbonne, France) at 2 μm resolution. Individual array images were acquired independently, adjusting the PMT gain for each image as recommended using the software Mapix® (Innopsys). Then, for each array image, raw expression data was extracted using the NimbleScan software v2.5 (Roche NimbleGen) and feature intensities were exported as .pair files.
Description
Complex environment. Enriched antisense mRNA. Total RNA extracted from groundwater from a site (site B) contaminated with chlorinated solvents (TCE), after a biostimulation treatment, in well P3.
Data processing
The background noise was determined using random probes present on the microarrays (5,707 probes in our experiment). This background noise is defined by two components: the background median intensity (Bposition) and its dispersion (Bdispersion). Finally, a modified signal-to-noise ratio called SNR’ and based on the formula of Verdik (Verdick, D., Handran, S. and Pickett, S. (2002) In LLC, D. P. (ed.), In G. Kamberova (ed.), DNA array image analysis: nuts and bolts. Salem, MA., pp. p. 83-98) is calculated as follow in order to reduce-centralize our data: SNR’ = (probe signal intensity - Bposition)/ Bdispersion. However, spatial effect across array surface is a predominant within-slide experimental artifact that needs to be eliminated before any other normalization procedure (Wang, X., He, H., Li, L., Chen, R., Deng, X.W. and Li, S. (2006) NMPP: a user-customized NimbleGen microarray data processing pipeline. Bioinformatics, 22, 2955-2957). Thus, for all array images obtained in this work, surface was segmented to 16 sub-squares according to probe position (X, Y) indicated in pair report. A Perl script was developed to calculate local background noise in all sub-squares and the median SNR’ retrieved from the eight replicates of each probe. Finally, another Perl script was implemented to summarize each replicate probe treated and determine the median value between the eight replicates. Positive hybridization was considered significant for probes having an SNR’ > 1.5 (value to avoid all false positives).
