Table 2.
Validation of microarray data using qRT-PCR of randomly selected genes relative to the housekeeping gene, rpoDa
| Locusb | Namec | Primer sequenced | Fragment (bp)e | Serovar Typhimurium Gene Functionf | Ratio of arcA mutant/WT | Log2 ratio | ||
|---|---|---|---|---|---|---|---|---|
| qRT-PCRg | Microarrayh | qRT-PCRi | Microarrayj | |||||
| STM3217 | aer | 5'-CGTACAACATCTTAATCGTAGC-3' 5'-TTCGTTCAGATCATTATTACCC-3' |
163 | aerotaxis sensor receptor, senses cellular redox state or proton motive force | 0.237 | 0.293 | -2.1 | -1.8 |
| STM1919 | cheM | 5'-GCCAATTTCAAAAATATGACG-3' 5'-GTCCAGAAACTGAATAAGTTCG-3' |
114 | methyl accepting chemotaxis protein II, aspartate sensor-receptor | 0.194 | 0.261 | -2.4 | -1.9 |
| STM0441 | cyoC | 5'-TATTTAGCTCCATTACCTACGG-3' 5'-GGAATTCATAGAGTTCCATCC-3' |
134 | cytochrome o ubiquinol oxidase subunit III | 4.920 | 5.465 | 2.3 | 2.5 |
| STM1803 | dadA | 5'-TAACCTTTCGCTTTAATACTCC-3' 5'-GATATCAACAATGCCTTTAAGC-3' |
155 | D-amino acid dehydrogenase subunit | 3.430 | 10.520 | 1.8 | 3.4 |
| STM2892 | invJ | 5'-TTGCTATCGTCTAAAAATAGGC-3' 5'-TTGATATTATCGTCAGAGATTCC-3' |
128 | surface presentation of antigens; secretory proteins | 0.855 | 1.010 | -0.2 | 0.0 |
| STM2324 | nuoF | 5'-GGATATCGAGACACTTGAGC-3' 5'-GATTAAATGGGTATTACTGAACG-3' |
163 | NADH dehydrogenase I chain F | 0.380 | 1.706 | -1.4 | 0.8 |
| STM0650 | STM0650 | 5'-CAACAGCTTATTGATTTAGTGG-3' 5'-CTAACGATTTTTCTTCAATGG-3' |
130 | putative hydrolase C-terminus | 0.274 | 0.123 | -1.9 | -3.0 |
| STM2787 | STM2787 | 5'-AAGCGAATACAGCTATGAACC-3' 5'-ATTAGCTTTTGCAGAACATGG-3' |
144 | tricarboxylic transport | 6.440 | 90.770 | 2.7 | 6.5 |
| STM4463 | STM4463 | 5'-AAGGTATCAGCCAGTCTACG-3' 5'-CGTATGGATAAGGATAAATTCG-3' |
142 | putative arginine repressor | 0.165 | 0.012 | -2.6 | -6.4 |
| STM2464 | eutN | 5'-AGGACAAATCGTATGTACCG-3' 5'-ACCAGCAGTACCCACTCTCC-3' |
153 | putative detox protein in ethanolamine utilization | 0.181 | 0.159 | -2.5 | -2.7 |
| STM2454 | eutR | 5'-GGTAAAAGAGCAGCATAAAGC-3' 5'-ATTATCACTCAAGACCTTACGC-3' |
118 | putative regulator ethanolamine operon (AraC/XylS family) | 0.189 | 0.188 | -2.4 | -2.4 |
| STM2470 | eutS | 5'-AATAAAGAACGCATTATTCAGG-3' 5'-GTTAAAGTCATAATGCCAATCG-3' |
137 | putative carboxysome structural protein, ethanol utilization | 0.197 | 0.105 | -2.3 | -3.3 |
| STM1172 | flgM | 5'-AGCGACATTAATATGGAACG-3' 5'-TTTACTCTGTAAGTAGCTCTGC-3' |
126 | anti-FliA (anti-sigma) factor; also known as RflB protein | 0.196 | 0.163 | -2.4 | -2.6 |
| STM3692 | lldP | 5'-TGATTAAACTCAAGCTGAAAGG-3' 5'-CCGAAATTTTATAGACAAAGACC-3' |
189 | LctP transporter, L-lactate permease | 5.950 | 12.780 | 2.6 | 3.7 |
| STM3693 | lldR | 5'-GAACAGAATATCGTGCAACC-3' 5'-GAGTCTGATTTTCTCTTTGTCG-3' |
153 | putative transcriptional regulator for lct operon (GntR family) | 5.750 | 80.000 | 2.5 | 6.3 |
| STM1923 | motA | 5'-GGTTATCGGTACAGTTTTCG-3' 5'-TAGATTTTGTGTATTTCGAACG-3' |
194 | proton conductor component of motor, torque generator | 0.282 | 0.253 | -1.8 | -2.0 |
| STM4277 | nrfA | 5'-GACTAACTCTCTGTCGAAAACC-3' 5'-ATTTTATGGTCGGTGTAGAGC-3' |
159 | nitrite reductase periplasmic cytochrome c(552) | 0.314 | 0.285 | -1.7 | -1.8 |
aSTM3211 (rpoD) is a housekeeping gene that was used as the reference gene where no significant
change in expression level was observed. The primer sequences (5' to 3') used for rpoD were as follows: CGATGTCTCTGAAGAAGTGC (forward) and TTCAACCATCTCTTTCTTCG (reverse). The size of the fragment generated is 150 bp.
bLocation of the open reading frame (ORF) in the S. Typhimurium LT2 genome.
cRespective gene name or symbol.
dFor each set, the first primer is the forward primer and the second primer is the reverse primer.
eSize of the amplified PCR product.
fFunctional classification according to the KEGG (Kyoto Encyclopedia of Genes and Genomes) database.
gExpression levels of quantitative reverse transcriptase polymerase chain reaction - values shown as the ratio between the arcA mutant and the wild-type; where values <1 indicate that ArcA acts as an activator, and values >1 indicate ArcA acts as a repressor.
hExpression levels from the microarray data - values shown as the ratio between the arcA mutant and the wild-type; where values <1 indicate that ArcA acts as an activator, and values >1 indicate ArcA acts as a repressor.
iExpression levels of quantitative reverse transcriptase polymerase chain reaction comparing the arcA mutant versus the wild-type - shown in signal to log2 ratio (SLR).
jExpression levels of microarray data comparing the arcA mutant versus the wild-type - shown in signal to log2 ratio (SLR).