Mrna specific primers

Total RNA from sus scrofa spleen tissue (preserved in our laboratory) was isolated using a TRIzol^® reagent kit (Takara Bio, Inc.) according to the manufacturer's instructions. cDNA synthesis was performed using a PrimeScript Reverse Transcriptase kit (Takara Bio, Inc.). Primers for the sus scrofa TFF3 gene were designed using Primer v.5.0 software (Sangon Biotech. Co. Ltd.), according to the gene sequence in the GenBank database (accession no. NM_001243483). mRNA specific primers (Sangon Biotech Co, Ltd.) were: TFF3 forward, 5′-GCATGGAGGCCAGGATGT-3′ and reverse, 5′-CGGTTAGAAGGTGCATTCT-3′. The PCR program to amplify the TFF3 gene from cDNA was 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 20 sec and 72°C for 30 sec with a final extension step at 72°C for 10 min. PCR products were separated by 2% agarose gel electrophoresis. Purified PCR fragments were retrieved using a PCR gel recovery kit (Takara Bio Inc.). Bands were visualized using the GelDoc XR⁺ (Bio-Rad Laboratories, Inc.) gel imaging system through nucleic acid staining. Densitometric analysis was performed using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Inc.).