First-strand complementary (c)DNA was generated in a 25-μl reaction volume containing 3 μg DNase I-treated total RNA, 400 pM oligo dT18, 0.4 mM dNTP, 20 U of an RNase inhibitor (Invitrogen), 100 U ReverTra BMS754807 Ace RTase (Toyobo, Tokyo, Japan), and 1× reverse-transcription (RT) buffer. The reaction was conducted at 42 °C for 1 h. After first-strand cDNA synthesis, a PCR of the housekeeping gene, elongation factor (EF)1α, was performed to check

the RT reaction. Transcripts of target genes (LGBP, PX, ppA, proPO I, proPO II, α2-M, integrin ß, HSP70, cytMnSOD, mtMnSOD, and ecCuZnSOD), and the internal control (EF1α) were measured by a qPCR. Primer sets for each gene were designed based on published L. vannamei genes using Beacon Designer Software vers. 6.0 ( Table 1). The recombinant plasmids containing LGBP, PX, ppA, proPO I, proPO II, α2-M, integrin ß, HSP70, cytMnSOD, mtMnSOD, and ecCuZnSOD qPCR

fragments were GDC 0199 all quantified to 1 μg μl−1. A series of concentrations of recombinant plasmids of 10−5–10−11 μg μl−1 was diluted with DEPC-treated water to construct the LGBP, PX, ppA, proPO I, proPO II, α2-M, integrin ß, HSP70, cytMnSOD, mtMnSOD, and ecCuZnSOD qPCR standard curves. Relationships between the threshold concentration (Ct) and copy number calculated based on the molecular weight of the target genes were established. Target gene expressions were quantified based on their relationships with the Ct

and copy number. All real-time PCRs used 10 μl 2× power SYBR GREEN PCR master mix (Applied Biosystems, Framingham, MA, USA) with 4 μl of sample, and 0.2 μM very each of the forward and reverse primers. Real-time PCRs were carried out on an ABI 7500 real-time PCR system (Applied Biosystems) using a program of denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 55–62 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min. After amplification, a melting-curve analysis was conducted to ensure that a single product was amplified. Each RNA sample and standard curve were examined in duplicate. A pathogenic strain of V. alginolyticus isolated from diseased L. vannamei, which displayed symptoms of anorexia, lethargy, and whitish musculature, was used for the study [ 3]. The bacterium was cultured in tryptic soy broth (TSB supplemented with 2% NaCl, Difco, Sparks, MD, USA) for 24 h at 28 °C, and then centrifuged at 7155g for 20 min at 4 °C [ 31]. The supernatant was removed, and the bacterial pellet was resuspended in a phosphate-buffered saline (PBS) solution at 1.9 × 108 colony-forming units (cfu) ml−1 as the bacterial suspension for the Vibrio challenge test. The WSSV inoculum and test solution were prepared based on a previously described method [27]. Briefly, 100 μl of haemolymph was withdrawn from WSSV-infected white shrimp and diluted with 400 μl of PBS.