qRT-PCR was performed as according to Appia-Ayme et al. (2012). Gene specific primers (~60°C Tm) were designed (2.4.21.2) to amplify an internal gene fragment of 95-100 bp (Table 20). Experiments were performed in biological triplicate with three technical replicates. Three 10-fold serial dilutions of chromosomal DNA (2.4.20.2) was used for standardisation. The calculated threshold cycle (Ct) for each gene was normalised to that of
reference genes rpoD and ampD; chosen as housekeeping genes whose expression levels remained constant across conditions tested. CFX manager software (BIO-RAD) was used for data analysis.
Cells were subjected to mRNA stabilisation and total RNA extraction (2.4.20). Following TurboDNAse (2U/ µL) treatment total RNA was subjected to quantity and quality analysis (2.4.20.2) and used as template for cDNA synthesis (2.4.21.1). Real-time PCR quantifications were realised on a 5-fold dilution of the total cDNA obtained, using the BIO-RAD CFX96™ instrument and SensiMix™ SYBR No-ROX kit (BIOLINE) (Table 7).
2.4.23.1 DNA synthesis
Total RNAs (2 µg) were reverse-transcribed from random hexamers (Invitrogen) with Superscript™ II Reverse Transcriptase (Invitrogen) according to manufacturer’s instruction. cDNA was diluted 5-times and stored long-term at -20˚C.
2.4.23.2 qRT-PCR primer design
qRT-PCR primers were designed using Primer3 (v. 0.4.0) primer design software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) with a product size
range of 95-105 bp and ‘General Primer Picking Conditions’ of; primer size 18-22 bp, GC content 50-60% and melting temperatures (Tm) of 58°C-62°C.
Primers were diluted 1:4 with nuclease-free dH2O to the 250 nM.
Table 20: Primers used during qRT-PCR to amplify 95-105 bp internal fragments of
target genes. rpoD and ampD were used as housekeeping reference genes.
Gene Forward Primer (5’-3’) Reverse Primer (5’-3’)
cueP (SL3616) CAGGGAGAAATGCCGAATAA CCGTTTCGATAGCTTTGCAT
baeR CGAAAGCCCGTTAATGATTG TACCCGGTTCAAGCGATAAG
cpxP TAACCGAACATCAGCGTCAG CCGATGCATTGTCTCCATTT
deoA ATTCATGCCAAAGACGAAGC ACGATAGACCGAAGGTGTGC
eco AAAGGAATGAAGCGGCAAGT CAATCCACATTCAGCGTTTG
rpoH ACGCTGATCCTGTCTCACCT TTGCCTTCCTGGATCAAATC
rpoN CAATGACGCCTCAGCTACAA TCAAGCAGCGGGTTATTTTC
scsA GCTCCTCCTCGACAGATGAC AAGGATAGCGCCTTCAAAGA
SL3009 CCTCTTCAGCAAACCCGTAA ATGCCATCCTGAGCTGATTT
SL3010 ATGAAATGTCACCGGACTGG ACGGGTTGTTACACGACCAT
SL3646 CATAAAATCGCGTCAACCAG CCATTCGTTCGAAGATCACA
SL4465 TCGATCGCCTGATTTTTACC AGCGCTTCTCCAGACGATAA
slrP ATATGCAGCGGCTAATGTCC ATGCAGTGGTCGAGTTACCC
spy GCCAATGATGCAGCATAAAG TAATGTCGCGAATTTGTTGC
stm3388 TCGATAGATGCGCCCTTTAC CATCAGTTCCCGTTCGTTTT
tsr CGGTGAAGTCCGTAATCTGG CAGCGTCGAACCTACATCAA
ybiJ ATGGCCTGGAAGCTAAACTG TGCCGCTCATTTTGTCATTA
yccA CGCGGTGTTTATCCTGATTT TGACGGTCGCACGAATATAG
ydgF AGCGTGCTGCTATTTGATGA TTACGCGTCCCTGATTTGAT
yedY TAACGCCGGAAGATAAGGTG CACGGTTCGGTTTTCAGACT
yihE GTTATCTTGGCCGATTGCAT GAATACCTGGCGAGGTTCAA
yjiY CCAACGCTGGCAGTAGGTAT AACAGCGCTTCGAACAGAAT
yncJ TACTGACCTGTGCGCTGTTT GGCTTCGTGTCTAAGCTGGT
yqaE GCTCGGCGTACTTTTAGGAA ACCCAAAATGCGTGGATAAG
zraP GCAACAATGACGGTATGTGG GCGCTGGTCTGCGTATAGTA
zraR GTTCGGTTAATTGCCGCTAC ATTTCAATCGCCACCACATT
Reference Genes
rpoD CGTACCCAGGAACGTCTGAT CAGGTTTCGCTGGTTTCATT
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2.4.24 Microarray
Total RNA samples were labelled with fluorescent dye Cy5-dCTP (Amersham) (2.4.23.1) and combined with SL1344 genomic DNA (reference), labelled with Cy3-dCTP dye using Gibco Bioprime DNA labelling system (2.4.23.2). Following hybridisation and incubation (2.4.23.3), array slides were scanned using a GenePix 4000A scanner (Axon Instruments, Inc.) and quantified using GenePix Pro 7.0 software (Axon Instruments, Inc.). Data was normalised using the Batch Anti Banana Algorithm in R (BABAR) (Alston et al., 2010) and further analysed using GeneSpring 7.3 Software (Agilent). As Cy dyes are light sensitive: reactions were kept in dark conditions during incubation steps and labelling.
2.4.24.1 Direct labelling of SL1344 reference genomic DNA
Samples were held on ice between incubation steps and labelled using the Gibco Bioprime DNA labelling system. The following protocol produces sufficient labelled gDNA for five hybridisations and was repeated twice to ensure sufficient labelled DNA for 8 x 15K array hybridisation.
Chromosomal DNA was isolated (2.4.20.2) and 2 µg brought to a volume of 21 µL in Sigma dH2O. Reaction buffer mix/2.5 x random primers from Gibco
Bioprime DNA labelling system was added (20 µL), samples incubated (100˚C, 5 min), followed by chilling on ice for 5 min. A dNTP mix (10x) (produced separately to the DNA labelling system [1.2 mM each dATP, dGTP, dTTP; 0.6 mM dCTP; 10 mM Tris, pH8.0; 1 mM EDTA]) was added (5 µL) on ice, followed by Cy3-dCTP (3 µL) and 1 unit of Klenow enzyme (1 µL) (Gibco Bioprime DNA labelling system). Samples were collected by brief centrifugation and incubated at 37˚C overnight.
Following labelling, unincorporated/quenched Cy dyes and residual reagents were removed (2.4.3), eluted twice in 50 µL Sigma H2O to maximize
recovery. Samples were stored in the dark, at 4˚C, for hybridisation later the same day. Samples should not be stored for prolonged periods and should be labelled as close to hybridisation as possible.
2.4.24.2 Array hybridisation and washing
To prevent impurities, 8 x 15K array slides should be handled in low dust environments while wearing clean, powder-free gloves. Hybridisations were carried out in 50 µL reactions (Table 21). Following labelling, all sample volumes were reduced from 100 µL to ~16 µL in a concentrator 5301 (Eppendorf®) at 30˚C. Labelled gDNA sample volumes were measured and
divided equally (1/5) between the labelled cDNA samples. Total volumes of combined samples were brought to 21.5 µL with Sigma H2O, and denatured
at 94˚C, 2 min. Following sample collection by brief centrifugation and cooling (RT, 1 min), labelled cDNA/gDNA samples were mixed with 37.5 µL of hybridisation buffer (Table 21). An Agilent SureHyb backing (gasket) slide was placed onto a SureHyb Microarray Hybridisation Chamber (Agilent) base and 40 µL of hybridisation mix pipetted onto the center of each segment on the gasket slide, one per array, ensuring the liquid did not touch the gasket silicone edges. An OGT array slide was lowered on top of the gasket slide (Agilent label face down) allowing the array surface to come into contact with the hybridisation mix. The chamber was sealed and secured with clamps, ensuring that the hybridisation mix was moving freely across the whole array surface. The array can be gently tapped upon a hard surface to loosen any bubbles that may have become attached to the gasket silicone edges. The array slide, in its assembly, was incubated at 55˚C for 60 hours, rotating horizontally, within a blacked-out hybridisation oven.
The array slide, post hybridisation, required washing before scanning could take place. Once released from the hybridisation chamber, the array slide was submerged in wash solution 1 (Appendix D) and the gasket slide removed. The microarray was washed in wash solution 1, with gentle mixing, at RT for 5 min. This washing was repeated in wash solution 2 (Appendix D) and the array was dried by centrifugation in an array microcentrifuge for 30 sec. This removed residual liquid without splashing or exposing the array to dust and impurity contaminants.
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2.4.24.3 Scanning, normalisation and data analysis
The dried microarray slide was inserted, array side (Agilent label) down, into a Genepix 4000A scanner (Axon Instruments, Inc.) and scanned according to the manufacturer’s instruction. Scanned images were filtered and quantified using Genepix Pro 7.0 (Axon Instruments, Inc.) software, allowing each spot to be aligned, normalised for signal intensity by removing any background fluorescence and the green/red (Cy3/Cy5) ratio calculated. Data were normalised using the Batch Anti Banana Algorithm in R (BABAR) (Alston et al., 2010) and analysed with GeneSpring 7.3 (Agilent) software, with a false discovery rate (FDR) of 0.05.