«(Über die Bedeutung der bakteriellen Genomplastizität für die Adaptation und Evolution asymptomatischer Bakteriurie (ABU) Escherichia coli ...»
Data analysis For each strain three independent hybridizations were performed. Mean values were calculated from the duplicate spots of the each gene in all three arrays. The mean of the normalized intensity values of the duplicate spots of each gene was used for further analysis. To avoid extreme intensity ratios for genes close to or below the detection limit, signal intensity values corresponding to a signal to noise (S/N) ratio 1.0 were scaled up to a value corresponding to an S/N ratio=1.0. ORFs were recorded as lacking/not detectable if the S/N ratio was below 1.0 in at least two of the three hybridization experiments. In addition, E. coli K-12 strain MG1655-specific ORFs were recorded as lacking/not detectable if the ratio of the individual S/N ratios of the analyzed strain and that of the reference strain MG1655 was 0.3 in at least two of three experiments. The missing/not detectable ORFs were then aligned with their chromosomal location to determine the number and the size of chromosomal regions absent in the different E. coli strain. In addition, the fact that the ORFs are arranged on the DNA macroarrays without regard to their chromosomal localization minimizes the record of false negative spots, at least with respect to regions consisting of more than one gene, because the probability
that two adjacent ORFs would be recorded as absent due to hybridization artifacts is very low. Multiple positive signals for single genes were verified by PCR.
Hierarchical cluster analysis of the hybridization data was performed with the CLUSTER software (Eisen et al., 1998) based on the presence or absence of genes. Red and black denote the presence or absence of ORFs, respectively. The output was displayed with the software TREEVIEW (Eisen et al., 1998).
4.2. Working with RNA
For RNA work, special care had to be taken in order to prevent contamination of the samples with exogenous RNases. Gloves were worn throughout the whole experiment and RNase free pipette tips and reaction tubes were used. For all buffers and solutions, water was pre-treated over night with 0.1 % (v/v) diethylpyrocarbonate (DEPC) at 37 °C and autoclaved twice to remove remaining DEPC.
4.2.1. Isolation of total RNA with RNAeasy Kit
RNA was isolated using the RNeasy Mini kit (QIAGEN) according to the supplied protocol.
All subsequent steps of the RNeasy protocol were performed at room temperature. For RNA isolation bacteria were grown at 37 °C in 125 ml of pooled human urine until the optical density OD600 reached 0.15. For RNA isolation from Luria Broth (LB) cultures, bacteria were grown without agitation at 37 °C in 25 ml until the OD600 reached 0.6. 50 ml of the urine culture and 4 ml of the LB culture were taken and centrifuged at 6000 rpm for 5 min. The supernatant was removed and pellets were resuspended in 20 ml (urine culture) and 4 ml (LB culture) of PBS and RNAprotect Qiagen 1:1 (v/v), respectively. Samples were incubated at room temperature (RT) for 5 min and centrifuged at 6000 rpm for 10 min. Bacterial pellets were either stored at -80 °C or bacterial RNA was immediately isolated. In the second case, bacterial pellets were resuspended thoroughly in 100 μl of lysozyme-containing TE buffer (50 mg/ml) and incubated at 37 °C for 5 s with vortexing every 2 min. The following steps of the protocol are consistent with those of the protocol supplied with the RNeasy Mini kit.
4.2.2. Removal of contaminating DNA by DNase treatment and RNA cleanup Contaminating DNA was removed from total RNA preparations by DNase I digestion. 15 μg RNA in a final volume of 85 μl were mixed with 10 μl 10 x DNase I buffer and 10 μl RNasefree DNase I (New England Biolabs). Samples were incubated for 1 h at 37 °C, followed by RNA cleanup using the RNeasy Mini kit (QIAGEN). According to the manufacturer’s instructions, 350 μl RLT buffer supplemented with 10 μl ß-mercaptoethanol and 250 μl 100 % (v/v) ethanol were added to the DNase-treated RNA samples, before loading them onto the purification columns. After brief centrifugation, the columns were transferred to fresh collection tubes and washed twice with 500 μl RPE buffer. Finally, RNA was eluted from the column in 30 μl nuclease-free water.
As a control for successful DNA removal, 2 μl of the DNase-treated RNA or 1 μl DNA as positive control were used as a template in a PCR reaction with primers binding within the coding sequence of the fimA gene. The DNase digest was considered as complete if no product could be amplified from the RNA samples.
4.2.3. Reverse transcription (RT) for cDNA synthesis
For cDNA synthesis, the Superscript III reverse transcription kit (Invitrogen) was used. 2 μg of total RNA in a final volume of 10 μl were mixed with 1 μg of random hexamer primers (Amersham Biosciences). Primer annealing was carried out at 65 °C for 5 min. After 5 min cooling, 9 μl of a reverse transcription mixture were added to the samples. The composition of
the RT-mix for 1 sample was:
1 μl 25 mM deoxynucleotide mix 1 μl 0.1 M dithiothreitol (DTT; kit component) 4 μl 5 x first strand buffer (kit component) 1 μl 40 U μl-1 RNase OUT recombinant RNase inhibitor (Invitrogen) 1 μl 200 U μl-1 Superscript III reverse transcriptase (kit component) cDNA synthesis was performed at 52 °C for 60 min, followed by heat inactivation of the transcriptase at 70 °C for 15 min.
4.2.4. Quantitative Real-Time PCR To evaluate expression of single genes, a quantitative Real-Time PCR (qRT-PCR) approach was used. This method employs polymerase chain reaction to amplify gene transcripts in presence of the SYBR Green I dye (BioRad). This fluorescent dye intercalates into double stranded DNA and emits signals collected by the optical camera within the MyiQ cycler (BioRad). The number of cycles needed to reach a certain fluorescent signal threshold (CT) was used to calculate transcript levels. Primers for selected genes were designed using the FastPCR software (Ruslan Kalendar, Institute for Biotechnology, University of Helsinki, Finland) with the following parameters: product length range from 190 to 300 nt; annealing temperature 57 - 59 °C. Before their use for expression profiling, the different primer pairs were checked for amplification efficiency with pooled cDNA from different experiments.
Only primer pairs with an amplification efficiency of at least 90 % were used. For copy
number estimation, cDNA samples derived from the reverse transcription reaction were 100fold diluted in dH2O and the reaction mix was prepared (for one reaction) as follows:
4.2.5. Expression profiling using DNA arrays Expression profiling is a technique to study the relative amounts of all transcripts at a given time of sample collection, thereby allowing to monitor the expression level of every single gene detectable by the array.
Array Layout For expression profiling oligonucleotide glass microarrays (Operon Biotechnologies, Inc.) were used. A single Operon E. coli Custom 55156017 array contains 10816 longmer oligonucleotide probes covering the complete genomes of six Escherichia coli strains (6 genomes and four plasmids). The number of open reading frames (ORFs) or genes represented is as follows: 4269 ORFs of non-pathogenic E. coli K-12strain MG1655, 5306 ORFs of enterohemorrhagic E. coli O157:H7 strain EDL933, 5251 ORFs of enterohemorrhagic E. coli O157:H7 strain Sakai, 5366 ORFs of uropathogenic E. coli strain CFT073, 322 ORFs of uropathogenic E. coli strain 536, 448 ORFs of uropathogenic E. coli strain UTI89, 3 genes of EHEC plasmid OSAK1, 10 genes of EHEC plasmid pO157_Sakai, 97 genes of EHEC plasmid pO157_EDL933 and genes of UPEC plasmid pUTI89. In addition, the array comprises also a number of positive and negative controls. Each probe contains an amino linker at the 5' end. Probes are spotted as single spots in 32 blogs (4 columns, 8 rows), each blog with 18 columns x 19 rows.
RNA isolation and cDNA labelling
Total RNA was prepared from mid-log phase cultures grown in urine at 37 °C followed by DNase treatment, as described in section 4.2.2. All procedures involving fluorescent dyes had to be done quickly and by avoiding exposure to light because of photosensivity. All solutions were prepared with DEPC-treated water. All experiments were done in triplicates including the overnight culture.
Reverse transcription was performed using SuperScript IIITM reverse transcriptase (Invitrogen) and the fluorescently labelled nucleotides Cy3- and Cy5-dCTP (Amersham Pharmacia, Freiburg, Germany). For primer annealing, 10 µg total RNA were mixed with 1 µg of hexamer oligos in a total volume of 15 µl. The annealing mix was heated for 10 minutes at 70 °C, then cooled down to room temperature for 5 min followed by brief centrifugation. In
the meantime, the reaction mix was prepared:
Then, 22 µl of the reaction mix was pipetted into a small PCR reaction tube together with 15 µl annealing mix and 4 µl Cy3- or Cy5-dCTP (1 mM) was added. The total mix 41 µl was incubated for 1h at 46 °C in the thermoblock of a thermocycler. After 25 min, another 1 µl of SuperScript IIITM reverse transcriptase (200 U/µl) was added. The reaction was stopped by addition of 5 µl EDTA (500 mM) and to hydrolyze RNA, 10 µl NaOH (1 M) was added, followed by incubation at 65 °C for 15 min. The reaction mixture was cooled down to room temperature and 25 µl Tris-HCl (1M, pH 7.5) was added.
Labelled targets were purified using the Qiaquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions with minor changes: Briefly, 5 volumes of PB buffer were added, the sample applied to column and centrifuged at max speed for 30 s. The column was then washed with 700 µl PE buffer and dried by centrifugation. cDNA was eluted in 30 µl dH2O. Of this, 1 µl was taken for quality control and quantification using the NanoDrop photometer. The remaining cDNA was dried using a vaccum centrifuge. The cDNA pellet was resuspended in 2 µl dH20.
Arrays were cleaned by compressed air from dust particles and pre-hybridised in pre-warmed OPArray Pre-Hyb solution at 42 °C for 1 h. During that time Wash Solution 1 was prepared by diluting OpArray Wash B 1:40 (v/v) with chromatography grade H2O. Arrays were washed for 5 min at 20-25 °C and immediately transferred to a box with sterile H2O, rinsed for 30 seconds. This step was repeated twice. The slides were dried in Falcon tubes with a hole in the bottom by centrifugation at 1200 rpm for 10 min. Residual liquid was removed by compressed air.
Array hybridisation The hybridization chamber was rinsed with sterile dH2O and dried thoroughly. In the four corners of the chamber, 15 µl of sterile dH2O were added in order to keep the humidity during hybridisation time. The OpArray was placed into the chamber with the DNA side up (barcode side up) and the spotted area was covered with a LifterSlip. Cy5- and Cy3-labelled targets were mixed with 36 µl of OpArray Hyb Buffer, denatured at 65 °C for 5 min and then applied slowly to one end of the LifterSlip in order to disperse across the OpArray surface.
The hybridization chamber was closed and the arrays were incubated in a water bath at 42 °C for 14-16 hours.
For Post-Hybridization washing following solutions were used:
After hybridisation was completed, arrays were washed in pre-warmed Wash Solution 2 at 42 °C for 10 min, transferred to Wash Solution 3 and shaken for another 10 min at RT.
Subsequently, arrays were washed twice in Wash Solution 4 at RT for 5 min. Alike in the pre
hybridisation step, drying was done by centrifugation of the array in Falcon tubes for 10 min and then the arrays were immediately scanned.
Scanning Hybridised and washed slides were scanned using a GenePix Model 4000B Microarray Scanner (Axon Instruments Inc., Union City, CA 94587, USA) with a resolution of 5 μm pixel size. The excitation frequencies of the two lasers were 532 nm and 635 nm. The gain settings for the photomultiplier tubes were adjusted to use the entire dynamic range of the instrument and to get comparable fluorescence yields in both channels. Images of Cy3 and Cy5 signals were recorded as 2 layer 16bit TIFF files and analysed using the GenePix Pro 6.0 software.
For each experiment, at least three independent hybridizations were performed, one with a dye switch. After removal of bad quality spots (if less than 70 % of foreground pixels were below background intensity plus 2 standard deviations in both channels or if the signal to noise ratio were below 3 in both channels or if the difference between ratio of medians and regression ratio exceeded 20 % in one of the channels) the remaining intensities were saved as gpr output data files. For statistical validation and further analysis the Acuity 4.0 (Molecular Devices, USA) software was used. For all data, the local background was subtracted from the intensity values of each spot on the array and normalized by both linear ratio-based methods and non-linear lowess including print-tip groups. For statistical significance, one sample t-test was applied and the resulting data set was exported to Microsoft Excel. Hierarchical clustering of genes for visualisation of expression patterns was performed with the CLUSTER software (Eisen et al., 1998). The output was displayed with the software TREEVIEW (Eisen et al., 1998).
For data analysis, a cut-off value of 1.7 was used although the commonly used threshold value is twofold (DeRisi et al., 1997; Wildsmith and Elcock, 2001). Several studies have shown that a lower cut-off ranging from 1.4 to 1.74 can be used reliably if the results are reproducible in more replicates (Perez-Amador et al., 2001).