Quantitative RT-PCR (qRT-PCR)
Total RNAs from tissues were extracted with RNAiso Plus (Takara)
according to the manufacturer’s instructions. Reverse transcriptions
were completed using a PrimeScriptTM RT reagent Kit
(Takara). qRT-PCR was performed by using a SYBR Green qPCR Kit (Takara,
CN) in the Applied Biosystems 7500 Fast Real-Time PCR System (Life
Technologies). PEDV load was determined by detecting the viral
nucleocapsid (N) gene (Fwd: 5’-CACCTCCTGCTTCACGTACA-3’ and
Rev:5’-AGCTCCACGACCCTGGTTAT-3’). The glyceraldehyde 3-phosphate
dehydrogenase (GAPDH, Fwd: 5’-TCATCATCTCTGCCCCTTCT-3’ and Rev: 5’-
GTCATGAGTCCCTCCACGAT-3’) was used as the reference gene. The relative
levels of viral RNA were calculated by using the
2-ΔΔCT method.