TABLE 2.
Oligonucleotide primers and conditions for RT-PCR analysisa
| Gene | Primer | Sequence | Size of PCR product (bp) | RNA per reaction (ng) | No. of PCR cycles |
|---|---|---|---|---|---|
| sca | F | 5′-AACCTCTGCCCGAAGATGTC-3′ | 540 | 250 | 25 |
| R | 5′-GATGGGACTTTGGTCTTGCG-3′ | ||||
| fruA | F | 5′-ACTCTATCGCGGTCAGTATCATTATT-3′ | 1,010 | 250 | 25 |
| R | 5′-TCTTCTCACTAACTTCCACGTCTTTA-3′ | ||||
| gor | F | 5′-CTATCCCTTCTATTCCTGGCTCTG-3′ | 910 | 200 | 30 |
| R | 5′-CGTGAACGAACTCTTCTGATCC-3′ | ||||
| spo768 | F | 5′-ACATTTTCTACCAATGCTTTCTCG-3′ | 1,124 | 200 | 30 |
| R | 5′-AAAAGAAGCCAATCGTCACTTTGT-3′ | ||||
| gtfG | F | 5′-TACTAATAGTCTGAATGACCGCTCTG-3′ | 903 | 400 | 50 |
| R | 5′-TCAAGAGCAGGACTAGATTCATAAAC-3′ | ||||
| prmA | F | 5′-AAGCGACACTGGATGAGAAGATTAT-3′ | 667 | 200 | 25 |
| R | 5′-GTGACTTGATCGTCCTCTTTGAGTC-3′ | ||||
| abc-nbd | F | 5′-CCGGATTTGAGAAAATATCGAG-3′ | 2,112 | 250 | 25 |
| R | 5′-TCGCCTTATTTATGGGGAGGA-3′ | ||||
| accA | F | 5′-AAAAATCACTCGTTTGTTTGAGTATG-3′ | 1,296 | 300 | 50 |
| R | 5′-TTTTTGTCTATTTTATAGTCTATCTCC-3′ | ||||
| ylbN-like | F | 5′-CCGGAGGTATATTCAAAAGGATATT-3′ | 677 | 250 | 30 |
| R | 5′-ATGCCAGCTGCAACTGTAAATTC-3′ | ||||
| Rgg | F | 5′-CTTATCGTAAAGTCGTCGGGAAAA-3′ | 905 | 250 | 30 |
| R | 5′-TTTCAGAGGAGGTTCTATTCTATT-3′ | ||||
| lacD | F | 5′-AAACGCTTGCCAGACTGCTT-3′ | 572 | 175 | 30 |
| R | 5′-ACCATCTTTGATGTAGGCTTCAACT-3′ | ||||
| proWX | F | 5′-GCAGGATGGAGAGATTCGTC-3′ | 595 | 250 | 50 |
| R | 5′-AAGATAAAAGAACCCAGTCC-3′ | ||||
| tnpA | F | 5′-CTTGCGTAGCCAGATGTCTAAAGT-3′ | 490 | 200 | 50 |
| R | 5′-TTTATGCTTCCGGCTCGTATGT-3′ |
a
Forward (F) and reverse (R) primers were used to detect mRNA expression of the indicated genes by RT-PCR (results are shown in Fig. 5). The amount of total RNA added to each RT-PCR reaction mixture and the number of cycles were optimized for each gene to ensure amplification within the liner range. The annealing temperature was 50°C in all cases.