Growth conditions and RNA isolation
Synechocystis PCC6803 cultures were grown autotrophically in BG-11 medium essentially as described by Williams [9]. Specifically, 500 mL cultures were grown in flat (4 cm across the light path) Bellco tissue culture flasks at 30°C under white light fluorescent illumination (Cool White® , General Electric) with an incident intensity of 80 μmoles photons m-2 s-1 . Aeration was provided by bubbling with air enriched with 3% CO2 at a rate of 300 mL min-1 . RNA was isolated using hot phenol conjunction with vigorous agitation with glass beads as described by Bhaya et al. [10].
Chimeric oligonucleotide design and PCR amplification
Figure 1 provides a flow chart of the preparation of the full-genome Synechocystis microarray. Oligonucleotides, synthesized by Sigma Genosys http://www.sigmagenosys.com/, were designed to amplify the complete coding region of each open reading frame from the sequenced genome of Synechocystis PCC6803. The only exceptions were instances for genes with a predicted length extending beyond 2 kb. Of the 3,168 open reading frames, fewer than 10% (261) of the genes were truncated at the 3'-end to the 2 kb limit. Each of the oligos is a chimeric sequence designed for first round synthesis and, as such, contained two components: a 5' universal sequence and a 3' gene-specific sequence as shown in Figure 2. The gene-specific region of the oligonucleotides begins at the first or last base of the open reading frame and continues inside until the Tm of the oligo was in the range of 58–62°C (25–45 nt, including the universal sequence). An algorithm (Sigma Genosys) based upon Primer3 (Whitehead Institute) was used for this purpose and resulted in a high frequency of successful amplification as discussed below. Importantly, the universal sequences were designed to include SapI restriction sites in order to facilitate downstream applications, including cloning into an expression vector. Aliquots of the oligonucleotides were transferred and mixed as primer pairs to separate locations of 96-well plates prior to delivery from the commercial source, which facilitated amplification in the 96-well format. The remaining products from the synthesis reaction were stored in individual tubes at -20°C.
First round amplifications were performed in 96-well microtiter dish format using PTC-100 Thermocyclers from MJ Research Inc. Five μ• of 20 μM premixed primer pairs were transferred to 0.2 ml Thermo-Fast® 96-well plates (Marsh cat. No. AB-0600) and mixed with 95 μl of Reaction mix 1 (For 110 reactions; 55 μl of 500 μg/ml chromosomal DNA, 1.1 ml Pfx Buffer® , 1.1 ml Pfx Enhancer reagent® , 220 μl 50 mM MgSO4, 165 μl 10 mM premixed dNTPs [Amersham Pharmacia cat. No. 93-77212], 88 μl Pfx DNA Polymerase® [Invitrogen cat. No. 11708-039] and 7.722 ml sterile, deionized water). Plates were sealed using Microseal™ 'A' film (MJ Research cat. No. MSA-50001) during thermal cycling. Step-down thermo-cycling conditions consisted of 2 min 94°C initial denaturation followed by ten rounds of 94°C for 30 s, 62°C(-1°C/cycle) for 45 s, and 68°C for 3 min. The step down cycling was followed immediately by 24 cycles of 94°C for 30 s, 52°C for 45 s and 68°C for 3 min. Five μl of each sample was mixed with an equal volume of 20% glycerol and loaded on 0.7% agarose/TAE gels stained with ethidium bromide and separated by electrophoresis in Bio-Rad Sub-cell® model 192 units. Gels were analyzed using an Alpha Imager™ 2000 workstation and software for image capture and Total Lab v. 1.10 (Phoretix) software for image analysis. Second round products were generated with universal primers and products of the first round of amplification. Specifically, 5 μl of the original 100 μl reaction was transferred to new plates and mixed with 95 μl reaction mix 2 (For 110 reactions; 1.1 ml 10X Buffer, 220 μl 50 mM MgSO4, 165 μl premixed 10 mM dNTP's, 550 μl 20 μM universal primer pair, 1.1 ml Pfx Enhancer reagent, 88 μl Pfx enzyme, and 7.227 ml sterile, deionized water). Amplifications were carried out by denaturation for 2 min at 94°C followed by 35 cycles of the following conditions: 94°C for 15 s, 55°C for 45 s, and 68°C for 2 min. After completion of all 35 cycles, an additional extension period at 68°C for 20 min was added to polish the ends. Five μl of each sample was loaded into 0.7% agarose gels and analyzed as described above. Oligonucleotides for unsuccessful products after the second round reaction were redesigned, resynthesized, and used for reamplification. PCR fragments were compared to a known size marker (Cat. No. 10068-013, Invitrogen). Products were scored successful if their length, as defined by gel analysis, was +/- 15% of their predicted length, contained only a single band, and were of adequate density.
Purification, preparation, and printing DNAs
Upon verification of successful amplification by gel analysis, each plate of second round PCR products was purified using Multiscreen-PCR plates from Millipore according to the manufacturers specifications, except that a pre-wash was incorporated to remove potential surfactant type agents present in at least some batches of the filtration units. The resulting products were resuspended in 50 μl sterile deionized water at room temperature on a rotary shaker for 30 minutes. The yield of each product was measured by calculating concentration from A260 values using Corning Incorporated's Costar UV transparent 96 well plates with the Molecular Devices Spectramax 384 Plus spectrophotometer. The products were then dried in a Savant DNA110 Speed Vac. Using a Qiagen Bio-Robot 3000™, each product was resuspended in water to 1.0 μg.μl-1 . To each sample, an equal volume of 2X Microspotting solution (Cat. No. MSS-1, Telechem) was added. Ten microliter of each product was transferred to two sets of 384 Micro Array plates (Cat. No. X7020, Genetix Limited). Using a Omnigrid arrayer (Gene Machines) and 16 Microquill 2000 pins (Majer Precision), arrays were printed in triplicate on Superamine slides (cat. No. SMM-25, Telechem), using 250 micron spacing, in 18 × 15 spot subgrids. To improve spot morphology, blotting was increased to 30 spots per dip, and pins were re-dipped into the sample after every 50 slides. Cross-contamination between successively spotted samples is a potentially severe problem and was found to be minimized by increasing the stringency of pin washing procedures beyond the manufacturer's recommendations by increasing sonication in the washing solution for 5 seconds at the highest power setting. Relative humidity was maintained at 50% and temperature at 23°C throughout the arraying procedure.
Array process verification
At the end of printing, one or two slides were used to verify the quality of printing such as spot morphology and intensity. Slides were baked at 80°C for one hour, washed with 0.1% SDS and rinsed in deionized water. The slide was then stained with 100 nanomole aqueous Syto-61 (Molecular Probes) solution, washed twice with 0.1% SDS, rinsed once in deionized water and scanned for fluorescence at 550 nm in the ScanArray 3000. Each wash or rinse was carried out at room temperature for 5 minutes. The scanned images were analyzed for spot morphology and intensity and the results of the analysis were used to optimize the printing process during each run.
Labeled cDNA production
Fluorescently labeled cDNA was produced using a two-step procedure involving cDNA production from target RNA using a reverse transcriptase reaction incorporating aminoallyl-modified deoxynucleotide (aadUTP), followed by the second step involving chemical coupling of fluorescent dye (either Cy3 or Cy5) to the introduced amino moieties of the newly synthesized cDNA. The cDNA was synthesized from 16 μg total RNA using 5 μg random 8-base oligonucleotides (Sigma GenoSys) and Superscript II™ reverse transcriptase (Invitrogen). RNA samples (control and experimental) were each mixed with random octamers in a total volume of 25 μl DEPC (diethylpyrocarbonate) treated water, heated to 65°C for 5 minutes and then transferred to 42°C. Subsequently, 25 μl of a premix containing 2 μl of 100 mM DTT (dithiothreitol), 10 μl 5X Superscript II buffer, 1 μl 50X aadNTP mix (25 mM dA, C, and GTP, 8.4 mM dTTP, and 16.6 mM aadUTP [Sigma Cat. No.A-5660]), 10 μl DEPC treated water, and 2 μl Superscript II was added to each tube. Following two hour incubation at 42°C, 4 μl 50 mM EDTA and 2 μl 10 N NaOH was added to each tube and incubated at 65°C for 20 minutes to degrade RNA. The reaction was neutralized by the addition of 4 μl 5 M acetic acid. The cDNA was purified using Millipore Microcon 30 centrifugal filter devices by diluting the reaction mix with sterile deionized water to a volume of 500 μl according to the manufacturers specifications. Each of the two samples was washed five times using 500 μl deionized water and the final retentate adjusted to approximately 50 μl to facilitate handling. The resultant solutions were dried at room temperature in the Speed Vac, and the pellets were resuspended in 20 μl 0.1 M Na2CO3 (pH 9.0) and mixed with 10 μl of previously prepared Cy3 or Cy5 dye. Preparation of the dyes was performed in advance and involved dissolving one tube of powdered Cy3 or Cy5 dye from Amersham Pharmacia Biotech Cat. No. PA23001, or PA 25001 respectively, in 55 μl dimethyl sulfoxide and storing at -20°C until use. The coupling reaction was carried out at room temperature in the dark for 1 hour. The reaction was quenched by the addition of 4.5 μl 4 M hydroxylamine, followed by incubation for an additional 5 min. The Cy3- or Cy5 dye-coupled cDNA samples were combined and purified using a Qiagen PCR product purification kit according to the manufacturer's specifications. Samples were adjusted to 14.75 μl using SpeedVac and remainder of the hybridization components containing 2.5 μl of 10 μg/μl salmon sperm DNA, 8.75 μl 20X SSC, 0.25 μl 10% SDS, and 8.75 μl formamide were added. The mixture was then heated for 2 minutes at 99°C, briefly centrifuged at high speed, and maintained at 42°C until the hybridization with the DNA microarray was begun.
Hybridization and wash conditions
Printed slides were baked at 80°C for 1 hour, washed twice for 2 minutes each at room temperature in 0.1% SDS and once in deionized water to remove unbound material. The slides were boiled in deionized water for 3 minutes to denature the printed DNA, dried using low speed centrifugation in a specialized microscope slide-accommodating rotor (Telechem). The DNA on the microarray was then subjected to a UV-cross-linking at a dose of 150 mJ/cm2 . Prehybridization was performed by incubating the slide in a fresh mixture of 100 ml of 25% formamide, 5X SSC, 0.1% SDS, and 1% BSA for 45 min. at 42°C in a Coplin jar. The slide was then rinsed under distilled water and dried using low speed centrifugation.
The slide was preheated at 42°C in a Telechem hybridization chamber by placing in a static 42°C incubator. The pre-warmed sample was pipetted and spread uniformly onto a 24 × 60 mm glass cover-slip (Fisher Scientific, Cat. No., 12-548-5P) and the pre-warmed slide was inverted and placed with the arrayed surface contacting the sample on the cover-slip surface. Deionized water (10 μl) was added to each of the reservoirs of a Telechem Hybridization Chamber, and the slide was transferred to and enclosed within the chamber. The slide was incubated in a static incubator at 42°C for 12–16 h, and washed by placing in a 250 ml solution of 2X SSC and 0.1% SDS at 42°C for 5 minutes with gentle agitation provided by rotation of a magnetic stir bar. The slide was transferred quickly to a solution of 0.1X SSC, and 0.1% SDS, incubated for 10 minutes at room temperature with gentle agitation, and washed 5 additional times in 0.1X SSC for 1 min. at room temperature. The slide was then rinsed briefly with deionized water and dried by low speed centrifugation. The slides were stored in the dark until scanned.