All other pig-specific forward (FOR) and reverse (REV) primer sequences, annealing temperatures, and product sizes are listed.
Specific forward primers for the miR-145 and the small nucleolar RNA, C/D box 48 (SNORD48), also known as RNU48, were designed according to their published sequences (NCBI Reference Sequence: NR_029686.1 and NR_002745.1, respectively) and in silico specificity analysis.
Specific forward primers were used for miR-pgrn-1 (5'- TCGTTATTCTAGGCCATGTGTC-3'), miR-pgrn-9 (5'-TTGGATAGGCACTTACTCTGG-3'), miR-E1k (5'-TTGATGTGCAAGATCTTCTCAT-3'), miR-Scr (5'-TTGATAGAACCTTAGAGCATCG-3') and mouse U6 (5'-TGGCCCCTGCGCAAGGATG-3').
The ligase was inactivated by incubation of mixture at 65°C for 20 min. For the first vectorette PCR, we used a specific forward primer for amplification of the 3' portion of the 2nd MHC class IIB exon and a reverse primer for the 5' portion.
Specific forward primers for other anisakid species were also designed based on the nucleotide sequences of the ITS region.
Because of high similarity in the gloverin family, specific forward primers for Bmglv2 and Bmglv3 were designed, while a degenerate forward primer was designed for cloning Bmglv4, and common reverse primer was used for Bmglv 2 3 (Table S1).
The specific forward primer for RNU6B was 5′-ACGCAAATTCGTGAAGCGTT-3′.
Specific forward primer for frataxin is situated in the third exon: 5′-CATACACGTTTGAGGACTATGATGTCT-3′ and specific reverse primer situated in the fourth exon: 5′-TTTGGCGTCTGCTTGTTGATC-3′.
Subsequently, nested PCR was performed with specific forward primers for the 5' RLM-RACE adaptor and reverse primers from a known sequence of the Fgf-10 gene (data not shown).
The stem loop RT primer contains a target site for a universal reverse primer (5'-GTAGGATGCC GeneWorks-3', GeneWorks) and a target site for UniversalProbe Library (UPL) Probe #21 (Roche Diagnostics), which were used in subsequent cDNA amplification processes together with a specific forward primer for miR-3099 (5'-CGCGTAGGCT AGeneWorks-3', GeneWorks).
PCR was performed on the internal transcribed spacer 1 (ITS 1) using two genus specific forward primers for Entomophthora and Pandora, respectively: Ml2: 5′-GCAACGGATCATCATGTAA-3′ and PnCNf: 5′-TTTGGGTTTAAATAGAAGGTTGA-3′ and reverse primers Nu-5.8S-3′: 5′-ACTACGTTCTTCATCGATGA-3′ [ 10] and PnCNr: 5′-AGGCAAAGCCTAGAGCACTT-3′ (unpublished).
