Impromp 2 reverse transcription system

PmVRP15 transcript expression levels in different tissues were qualitatively assayed by reverse transcriptase-PCR (RT-PCR). The different tissues of ∼20 g body weight shrimps such as antennal gland, epipodite, eye stalk, gill, heart, hemocytes, hepatopancreas, intestine and lymphoid, were collected from uninfected shrimps, and then total RNA was extracted from each tissue using the TRI Reagent (Molecular Research Center). After DNase I (Fermentas) treatment, the total RNA (1 μg) was first converted to single-stranded (ss)cDNA with the ImPromp-II reverse transcription system (Promega) according to the manufacturer's instruction. Then, in the second stage, PmVRP15 transcript levels in each tissue were identified by PCR using 1 μL of the cDNA as a template with the PmVRP15F/R primers (Table 1). The EF-1α gene fragment was amplified using the EF-1-F/R primers (Table 1) as an internal control. The PCR thermal cycling conditions consisted of 94°C for 3 min, followed by 35 (for PmVRP15) or 27 (for EF-1α) cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s, and then a final extension at 72°C for 5 min. The PCR product was resolved by 1.5% (w/v) agarose-TBE gel electrophoresis and visualized by uv-transillumination following staining with ethidium bromide.