Tiling microarray design
The genomic tiling microarray probe set consisting of 385,000 oligonucleotide sequences was dynamically designed from both forward and reverse-complement strands of the target genome P. gingivalis strain W83, using a tiling array probe design algorithm developed by Høvik and Chen . The probe set can be downloaded from the Microbial Transcriptome Database http://bioinformatics.forsyth.org/mtd. The probes were printed on high-density microarrays by Roche NimbleGen, Inc. (Madison, WI, USA).
Bacterial culture preparation
P. gingivalis strain W83 was cultured anaerobically on trypticase soy agar (TSA) plates containing sheep blood, hemin, and vitamin K (BAPHK)  for 48 hours at 37°C. Upon harvest, a solution containing 2:1 (v:v) ratio of RNAprotect Bacteria Reagent (Qiagen, Valencia, CA, USA) and 1X PBS was poured onto the colonies and incubated anaerobically for 5 min. The cells were mixed and suspended in this solution, pelleted by centrifugation at 5,000 × g, 4°C for 10 min, and then subjected to either RNA or DNA extraction.
Total RNA extraction
Lysis of bacterial cells was performed according to the protocol provided with the MasterPure RNA Purification Kit (Epicentre, Madison, WI, USA). The lysate was treated with Proteinase K at 65°C for 15 min and placed on ice. To increase the recovery rate of small RNA, 0.1 volume of 5 M NaCl was added to the lysate. Acid-phenol: chloroform (5:1) (v:v) extraction was then done using Phase-Lock Gel Heavy tubes (Eppendorf, Hauppauge, NY, USA). Total RNA in the upper aqueous phase was purified with a solid-phase extraction filter supplied in the mirVana miRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX, USA) according to the recommended protocol. The filter-trapped RNA was washed and eluted with the provided wash solutions and elution buffer. To completely remove genomic DNA, the RNA extract was treated twice with Turbo DNase (Applied Biosystems/Ambion) at 37°C for 30 min, and purified again with the mirVana miRNA Isolation Kit.
RNA direct labeling and microarray hybridization
The Label IT Cy3 Reagent (Mirus Bio, Madison, WI, USA) was used to directly label total RNA. To optimize the RNA labeling efficiency, 1 μg RNA was mixed with 4 μl Label IT Reagent and incubated in a 100-μl final volume for 4 hours at 37°C. To improve hybridization efficiency, the labeled RNA was fragmented to an average size of 80-100 nucleotides with 0.25 volume of 5X Fragmentation Buffer (Mirus Bio) and incubated at 94°C for 15 min. The fragmented and fluorescently labeled RNA was purified with the mirVana miRNA Isolation Kit before hybridization.
RNA-DNA hybridization was performed on microarray slides covered with a HybriWell chamber (Grace Bio-Labs, Bend, OR, USA). Prehybridization was carried out for 90 min at 42°C in a 400 μl solution containing 343 μl Long Oligo hybridization buffer [94 mM Tris/HCl pH 7.0, 9.4 mM EDTA, 29.15% formamide, 5.83X SSC, 0.12% SDS], 0.5 mg/ml BSA, and 0.1 mg/ml salmon sperm DNA (Applied Biosystems/Ambion). The salmon sperm DNA was denatured at 95°C for 5 min before added to the prehybridization and hybridization solutions. After the prehybridization the HybriWell chamber was removed and the slide washed with nuclease-free water. The slide was then spin-dried at low speed for 2 min, and a new HybriWell chamber was sealed onto the slide. For each microarray 3 μg of labeled RNA was denatured at 65°C for 5 min in a final volume of 300 μl hybridization solution containing 257 μl Long Oligo hybridization buffer, 3 μl Alignment Oligo (NimbleGen), and 0.7 mg/ml salmon sperm DNA. Denatured RNA was then loaded in the chamber and hybridization was carried out at 42°C for 16 hours with 10 rpm rotation in an oven (Labnet, Edison, NJ, USA). The slide was washed according to NimbleGen's protocol (NimbleGen Arrays User's Guide Gene Expression Analysis).
cDNA labeling and microarray hybridization
Synthesis of cDNA and biotin end-labeling were performed according to NimbleGen's protocol (Prokaryotic Biotin-Label Procedure). First-strand cDNA was synthesized from 10 μg total RNA. The RNA together with 3 μg random hexamer primers (Invitrogen, Carlsbad, CA, USA) in a 12-μl volume was denatured at 70°C for 10 min. The solution was then cooled to 25°C and mixed to a final concentration of 0.5 mM dNTP, 20 mM DTT, 1X first strand buffer and 0.75 U/μl RNaseOUT (Invitrogen) followed by heating to 42°C. A total of 1200 U SuperScript II reverse transcriptase (Invitrogen) was added to a final volume of 54 μl and the mixture incubated overnight at 42°C. The cDNA product was treated with 0.02 U/μl RNase H (Invitrogen) and 0.01 μg/μl RNase A (Epicentre) in a 100-μl volume to eliminate RNA contaminants. The cDNA was then purified with standard procedures for phenol: chloroform extraction using Phase-Lock Gel Light tubes (Eppendorf) followed by ethanol precipitation. Purified cDNA was fragmented to 50-200 nucleotides in size with DNase I (Applied Biosystems/Ambion), and labeled at the 3'end in a 100-μl volume containing 0.5 U/μl Terminal Deoxynucleotidyl Transferase (Promega, Madison, WI, USA) and 0.025 mM Biotin-N6-ddATP (Enzo Life Sciences Inc., Farmingdale, NY, USA). The labeling reaction was carried out for 2 hours at 37°C. The biotin-labeled cDNA was then concentrated using Microcon YM-10 filters (Millipore, Billerica, MA, USA). Microarray hybridization was carried out with the customer service provided by NimbleGen.
Genomic DNA extraction and hybridization
Genomic DNA for DNA reference microarray hybridization was extracted with the MasterPure DNA Purification Kit (Epicentre) and RNA was removed using the RNase A supplied in the kit. Genomic DNA was fragmented with DNase I (Applied Biosystems/Ambion) to 100-200 nucleotides in size and then labeled either with biotin 3'end-labeling (i.e., cDNA procedure) or with the Label IT Reagent (i.e., direct RNA labeling). Conditions for microarray hybridization were the same as described in the previous sections for cDNA or RNA hybridization, respectively.
Data acquisition and normalization
After washing and drying, the microarray slides were immediately scanned in a GenePix 4000B Scanner (Axon Instruments, Union City, CA, USA) using the provided GenePix Pro 6.1 software. NimbleScan v2.5 software was then used for spot features extraction from the scanned images. Each of the microarray hybridization results hybridized with RNA, cDNA, or genomic DNA, consisted of at least two biological repeats. Two types of normalizations were performed - normalization by DNA reference array and between-array normalization. For DNA reference array normalization, intensities of cDNA and RNA signals were normalized with signals from DNA reference arrays that were hybridized with fragmented genomic DNA labeled in the corresponding way, i.e., biotin end-labeling and chemical labeling for cDNA and RNA, respectively. Both normalizations were done using the Bioconductor R package "tilingArray" . Coding and non-coding regions were determined based on the annotation of the P. gingivalis W83 genome available from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov). The baseline (i.e., background level) of each resulting intensity profile was calculated and represents the average value of the probe signal intensities from all intergenic regions. Intensities of probe sequences falling within 200 nucleotides to both ends of the intergenic sequences were excluded from the calculation to avoid possible positive signals from either 5'- or 3'end untranslated regions.
Strand-specific RT-PCR using tagged primers
In conventional reverse transcription PCR (RT-PCR), false positive PCR artifacts have been reported in absence of primer used in the reverse transcription step, due to self-priming of RNA or non-specific small DNA/RNA oligonucleotide contaminants that can also be used as primers in RT reactions . To avoid this problem, first-strand cDNA was synthesized with a genome specific primer attached with a tag sequence to the 5'end . The tag sequence was unique and not found in the genome of P. gingivalis. The subsequent PCR was then carried out with the tag sequence as one of the paired primers. Sequences of the primers used are listed in Table S1, Additional file 1. As a result, only the cDNA synthesized with the tagged primer can be amplified. A separate set of primers for amplifying the sense strand of one of the housekeeping genes in P. gingivalis - mutB, was included as positive control in all RT-PCRs. Negative controls were performed without the addition of reverse transcriptase to the RT reactions. For RT reactions, 2 μg RNA and 2 μl of both tagged and mutB RT-primers (2 μM) were mixed in a 12-μl volume, denatured by heating at 65°C for 5 min, and chilled on ice. To the RNA/primer solution the following reagents were added to a final concentration of 0.5 mM dNTP, 20 mM DTT, 1X first strand buffer, and 2 U/μl RNaseOUT (Invitrogen). The solution was heated to 50°C and mixed with 200 U of SuperScript III reverse transcriptase (Invitrogen) in a final volume of 20 μl. The mixture was incubated at 50°C for 50 min and the RT was terminated by heat inactivation for 5 min at 85°C. To remove RNA, RNase A (Epicentre) was added to a final concentration of 0.01 μg/μl and incubated for 10 min at 37°C. RT-primers were then removed using the MinElute PCR Purification Kit (Qiagen). PCR amplification was performed in a 20-μl volume containing 1.2 μl of the RT product, 17 μl Platinum Blue PCR SuperMix (Invitrogen), and 1 μl of each forward and reverse primers (10 μM). The thermal cycling conditions were: 2 min at 95°C followed by 25 cycles of 30 sec at 95°C, 30 sec at 55°C, and 20-90 sec (depending on PCR product length) at 72°C.