Standard curves created for all primers had a slope of -3.3 ± 0.3 (data not shown). For quantification of amplicons, an individual gene was first amplified by PCR and cloned into the pGEM-Teasy vector (Promega, Madison, WI). Recombinant plasmid DNA was then purified and diluted serially 10-fold to generate a standard curve. Transcript copies corresponding to each gene of interest were calculated Selleck AZD6244 using the Absolute Quantification Analysis program (Applied Biosystems) and normalized against copies of flaB. Table 1 Oligonucleotide primers

used in this study Gene Forward (5′-3′) Reverse (5′-3′) Cloning     flaB GGGAACTTGATTAGCCTGCGCAAT TCGAGCTTCTGATGATGCTGCT rpoS CTTGCAGGACAAATACAAAGAGGC TGGGACTATTGTCCAGGTTATATCTTT ospC CTGCTGATGAGTCTGTTAAAGGGC TTTGGACTTTCTGCCACAACAGGG ospA GCGTTTCAGTAGATTTGCCTGGTG

CCCTCTAATTTGGTGCCATTTGAGTCG dbpA CTTATATCATGTGGACTAACAGGAGC AGCACTCCTTGAGCTGTAGTTGGA qRT-PCR     flaB TGATTAGCCTGCGCAATCATT AATGACAGATGAGGTTGTAGCAGC rpoS CTGGACAAAGAAATAGAGGGATCTG CAAGGGTAATTTCAGGGTTAAAAGAA ospC GTACTAAAACTAAAGGTGCTGAAGAA GCATCTCTTTAGCTGCTTTTGACA ospA GGCGTAAAAGCTGACAAAAGTAAAGT TGTTTTGCCATCTTCTTTGAAAAC dbpA ACGAAGCGCTAAAGACATTACAGA GGCATCAAAATTTACGCCCTTA Acknowledgements We thank Jianli Zhou for A-769662 in vitro technical assistance. This work was supported by NIH-NIAID Grants AI-059062 (to MVN), AI-076705 (to SN) and AI-080615 (to UP), an Arthritis Foundation fellowship (to GN), and an award from the American Heart Association (to ZO). References 1. Bacon RM, Kugeler KJ, Mead Liothyronine Sodium PS: Surveillance for Lyme disease-United States, 1992–2006. MMWR Surveill Summ 2008,57(10):1–9.PubMed 2. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP: Lyme disease-a tick-borne spirochetosis? Science 1982,216(4552):1317–1319.PubMedCrossRef 3. Steere AC, Grodzicki RL, Kornblatt AN, Craft JE, Barbour AG, Burgdorfer W, Schmid GP, Johnson E, Malawista SE: The spirochetal etiology of Lyme disease. N Engl J Med 1983,308(13):733–740.PubMedCrossRef 4. de Silva AM, Telford SR, Brunet LR, Barthold SW, Fikrig E: Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine.

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