TempliPhi™ amplified DNA samples from field isolates were compared to control strain 232 using a two-color experimental microarray design. Independent samples from one isolate labeled with one dye were paired with control samples labeled with the alternate dye; the samples were mixed and hybridized to the microarray.
Growth protocol
All M. hyopneumoniae strains were grown in Friis media as previously described in [Friis, N. F. 1975. Some recommendations concerning primary isolation of Mycoplasma hyopneumoniae and Mycoplasma flocculare, a survey. Nord. Veterinaermed. 27:337-339] and are from in vitro passage less than 15. Cultures consisted of 125 ml of Friis media in 250 ml Erlenmeyer flasks incubated at 37°C with slow agitation until the culture reached mid log phase as indicated by color change and turbidity. Mycoplasmas were pelleted by centrifugation at 24,000 x g, and the cell pellets were stored at 70°C until the chromosomal DNA was isolated.
Extracted molecule
genomic DNA
Extraction protocol
DNA isolation. DNA was isolated from frozen cell pellets as follows. The cells were first resuspended in 1 ml of TNE buffer (10 mM Tris, 140 mM sodium chloride, 1 mM ethylenediamine tetraacetic acid, pH 8.0), and Proteinase K was added to a final concentration of 70 µg/ml. The suspension was incubated at 50°C for 5 min, and then sodium dodecyl sulfate was added to a final concentration of 0.1% and incubation was continued at 50°C for 4 h. The suspension was then extracted with an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol three times, and the DNA was precipitated by the addition of one tenth volume of 3 M sodium acetate and bringing the solution to 70% ethanol as described in [Sambrook, J., and W. R. David. 2001. Purification of nucleic acids, p. A8.9-A8.15. In J. Argentine (ed.), Molecular Cloning: a laboratory manual, Third ed, vol. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.]. The DNA pellets were dissolved in nuclease-free water, and samples were quantified and checked for purity using the Nanodrop® ND-1000 Spectrophotometer (Nanodrop, Wilmington, Del.). TempliPhi™ reactions. Field isolate samples yielded low amounts of genomic DNA compared to strain 232 due to their fastidious growth and lack of adaptation to growth media. To overcome the issue of limited quantities of DNA, genomic samples were amplified using the TempliPhi™ 100 Reaction Kit (Amersham, Biosciences, Piscataway, N.J.) according to the manufacturer’s protocol. A total of five reactions were combined for each field isolate and strain 232, yielding approximately 5-8 µg total DNA in each preparation which was subjected to mechanical shearing. Nebulization. The DNA was mechanically sheared prior to labeling to ensure an optimized fragment size for efficient labeling and hybridization. Each amplified sample was added to the modified nebulizer (product # 4100, MEDEX, Carlsbad, Calif.) containing 2 ml of sterile 50% glycerol. The nebulizer was modified by removing the plastic cuff, trimming the edge and inverting it during reassembly. The samples were sheared using a 10 psi nitrogen stream for 15 min. The fragment size of less than 1,000 base pairs was optimal for efficient labeling and signal strength. This was confirmed by gel electrophoresis on 1.5% agarose gel.
Label
Cy3
Label protocol
Targets were generated and purified from mechanically sheared DNA samples using the BioPrime® Plus Array CGH Indirect Genomic Labeling System (Invitrogen Corp., Carlsbad, Calif.). A set of 129 open reading frame-specific hexamer oligonucleotide primers was used to generate amino-allyl modified DNA targets. These targets were then labeled with either Alexa Fluor™ 555 Reactive Dye or Alexa Fluor™ 647 Reactive Dye (Molecular Probes, Inc.) according to the experimental design.
TempliPhi™ amplified DNA samples from field isolates were compared to control strain 232 using a two-color experimental microarray design. Independent samples from one isolate labeled with one dye were paired with control samples labeled with the alternate dye; the samples were mixed and hybridized to the microarray.
Growth protocol
All M. hyopneumoniae strains were grown in Friis media as previously described in [Friis, N. F. 1975. Some recommendations concerning primary isolation of Mycoplasma hyopneumoniae and Mycoplasma flocculare, a survey. Nord. Veterinaermed. 27:337-339] and are from in vitro passage less than 15. Cultures consisted of 125 ml of Friis media in 250 ml Erlenmeyer flasks incubated at 37°C with slow agitation until the culture reached mid log phase as indicated by color change and turbidity. Mycoplasmas were pelleted by centrifugation at 24,000 x g, and the cell pellets were stored at 70°C until the chromosomal DNA was isolated.
Extracted molecule
genomic DNA
Extraction protocol
DNA isolation. DNA was isolated from frozen cell pellets as follows. The cells were first resuspended in 1 ml of TNE buffer (10 mM Tris, 140 mM sodium chloride, 1 mM ethylenediamine tetraacetic acid, pH 8.0), and Proteinase K was added to a final concentration of 70 µg/ml. The suspension was incubated at 50°C for 5 min, and then sodium dodecyl sulfate was added to a final concentration of 0.1% and incubation was continued at 50°C for 4 h. The suspension was then extracted with an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol three times, and the DNA was precipitated by the addition of one tenth volume of 3 M sodium acetate and bringing the solution to 70% ethanol as described in [Sambrook, J., and W. R. David. 2001. Purification of nucleic acids, p. A8.9-A8.15. In J. Argentine (ed.), Molecular Cloning: a laboratory manual, Third ed, vol. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.]. The DNA pellets were dissolved in nuclease-free water, and samples were quantified and checked for purity using the Nanodrop® ND-1000 Spectrophotometer (Nanodrop, Wilmington, Del.). TempliPhi™ reactions. Field isolate samples yielded low amounts of genomic DNA compared to strain 232 due to their fastidious growth and lack of adaptation to growth media. To overcome the issue of limited quantities of DNA, genomic samples were amplified using the TempliPhi™ 100 Reaction Kit (Amersham, Biosciences, Piscataway, N.J.) according to the manufacturer’s protocol. A total of five reactions were combined for each field isolate and strain 232, yielding approximately 5-8 µg total DNA in each preparation which was subjected to mechanical shearing. Nebulization. The DNA was mechanically sheared prior to labeling to ensure an optimized fragment size for efficient labeling and hybridization. Each amplified sample was added to the modified nebulizer (product # 4100, MEDEX, Carlsbad, Calif.) containing 2 ml of sterile 50% glycerol. The nebulizer was modified by removing the plastic cuff, trimming the edge and inverting it during reassembly. The samples were sheared using a 10 psi nitrogen stream for 15 min. The fragment size of less than 1,000 base pairs was optimal for efficient labeling and signal strength. This was confirmed by gel electrophoresis on 1.5% agarose gel.
Label
Cy5
Label protocol
Targets were generated and purified from mechanically sheared DNA samples using the BioPrime® Plus Array CGH Indirect Genomic Labeling System (Invitrogen Corp., Carlsbad, Calif.). A set of 129 open reading frame-specific hexamer oligonucleotide primers was used to generate amino-allyl modified DNA targets. These targets were then labeled with either Alexa Fluor™ 555 Reactive Dye or Alexa Fluor™ 647 Reactive Dye (Molecular Probes, Inc.) according to the experimental design.
Hybridization protocol
Following purification of the fluorescently labeled cDNA per manufacturer’s instructions, samples were dried in a vacuum centrifuge and then resuspended in 10 µl Pronto! cDNA/long oligo hybridization solution (Corning). Targets were denatured at 95°C for 5 min and centrifuged at 13,000 x g for 2 min at room temperature. Labeled targets from one 232 control and one field isolate were then combined, pipetted to an array, and covered with a 22 x 22 mm HybriSlip™ (Schleicher & Schuell, Keene, N. H.). Slides were placed in a Corning hybridization chamber and incubated in a 42°C water bath for 12-16 h. Slides were washed according to Corning’s UltraGAPS™ protocol and dried by centrifugation.
Scan protocol
Eight of the fourteen isolate arrays (95MP1504, 95MP1505, 95MP1506, 95MP1507, 95MP1509, 95MP1510, 00MP1301, and 00MP1502) were scanned with each dye channel using a ScanArray Express laser scanner (Applied BioSystems, Inc., Foster City, Calif.) under varying laser power and PMT gain settings to increase the dynamic range of measurement. The other six arrays (95MP1501, 95MP1502, 95MP1503, 95MP1508, 97MP0001, 05MP2301) were scanned with an Applied Precision’s ArrayWoRx® Biochip Reader (Applied Precision, Inc., Issaquah, Wash.).
Description
White light scanner. Biological replicate 1 of 4.
Data processing
Images were analyzed for spots and signal intensities quantified using the softWorRx Tracker software package (Applied Precision, Inc.). Spot-specific mean signals were corrected for local background by subtracting spot-specific median background intensities. The natural logarithm of the background-corrected signals from a single scan were adjusted by an additive constant so that all scans of the same array-by-dye combination would have a common median. The median of these adjusted-log-background-corrected signals across multiple scans was then computed for each spot to obtain one value for each combination of spot, array, and dye channel. These data for the two dye channels on any given array were normalized using LOWESS normalization to adjust for intensity-dependent dye bias. Following LOWESS adjustment, the data from each channel were adjusted by an additive constant so that the median for any combination of array and dye would be the same for all array-by-dye combinations. The difference in normalized values for each spot was calculated as the signal intensity of Alexa 555 dye minus Alexa 647 dye. The differences for the triplicate spots were then averaged within each array to produce one normalized difference value for each of the 627 probe sequences.
