PMC:7640975 / 17368-19151
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7640975","sourcedb":"PMC","sourceid":"7640975","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7640975","text":"2.9 Quantitative Real-Time PCR (qPCR)\nTo verify the DEPs identified by iTRAQ at the transcriptional level, the mRNA of two representative DEPs, the downregulated ANAPC7 and the upregulated IFIT1, were detected by qPCR. Total cellular RNA of both PDCoV- and mock-infected IPEC-J2 cells was extracted using the TaKaRa MiniBEST universal RNA extraction kit (Dalian, China). The first strand cDNA was synthesized using 2 μg of cellular RNA and with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) following the manufacturer’s instructions. The qPCR assays were conducted using a TaKaRa SYBR Premix Ex Taq kit on an ABI 7500 real-time PCR machine along with β-actin as a housekeeping gene. The primers used for the qPCR assay were designed based on the NCBI reference sequences for ANAPC7, IFIT1 and β-actin genes under GenBank accession numbers NM_016238, HQ679904 and NM_001101, respectively. The information for the designed primers was as following: ANAPC7 (Forward: 5′-CTTTGCTGAGGAACGCACTG-3′, Reverse: 5′-TCCATGTCGTCCACATCCTC-3′); IFIT1 (Forward: 5′-GAAGATTTAACCCAACAAGAACATA-3′, Reverse: 5′-CTTTCGATACGTAAGGTAATACAGC-3′); β-actin (Forward: 5′-TCCCTGGAGAAGAGCTACGA-3′, Reverse: 5′-AGCACTGTGTTGGCGTACAG-3′). The qPCR assays were carried out in a 20-μL reaction volume comprised 10 μL of TaKaRa SYBR Premix Ex Taq, 0.4 μL of each forward and reverse primer (10 μM), 7.2 μL of nuclease-free water, and 2 μL of template. The qPCR amplifications were carried out using a thermal profile with an initial step at 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s (for ANAPC7 and β-actin) or 55 °C for 10 s (for IFIT1) and 72 °C for 40 s. Fold-change values were calculated in term of the 2–ΔΔCt method and normalized to the β-actin reference 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