Abi prism 7900ht sequencing detection system

Stem-loop qRT-PCR was used to confirm the differential expression of ssc-miR-10b, ssc-miR-30a-5p, ssc-miR-21, and ssc-let-7f in Fa wild-type strain0infected PK-15 cells, and ssc-miR-182, ssc-miR-192, ssc-miR-19b, and ssc-miR-24-3p in PRV Fa ΔgE/gI strain-infected PK-15 cells. The expression levels of PRV-encoded ssc-miR-novel-chrPRV_425, ssc-miR-novel-chrPRV_428, ssc-miR-novel-chrPRV_434, ssc-miR-novel-chrPRV_435, and ssc-miR-novel-chrPRV_441 were also confirmed by stem-loop qRT-PCR. Experiments were performed in triplicate on the ABI Prism 7900HT sequencing detection system (Applied Biosystems, Foster City, CA, USA). Data analyses were performed using a two-tailed Student’s t test.

cDNA was first synthesized using equivalent amounts of total RNA (0.5-1 μg) with random primers in a 20 μl reverse-transcriptase reaction mixture (Promega). Real-time quantitative RT-PCR (Taqman) primers for snail1, snail2, twist, vimentin, E-cadherin, and β-actin were designed and purchased from Applied Biosystems as Assay-on-Demand™ Gene Expression Products. Real-time RT-PCR was performed following the supplier’s directions. The 20 μl PCR mixture contained 10 μl of 2× Taqman universal PCR master mixes, 1 μl of 20× working stock of expression assay mix, and 50 ng RNA converted DNA. Real-time PCR was performed in an ABI PRISM 7900HT sequencing detection system (Applied Biosystems, Foster City, CA, USA). The assay for each sample was performed in triplicate. Fluorescence of the PCR products was detected using same apparatus. The number of cycles required for the amplification plot to reach the threshold limit, the Ct value, was used for quantification.