Cross-Species Genome-Wide Analysis Reveals Molecular and Functional Diversity of the Unconventional Interferon-? Subtype

Shields, Lauren E.; Jennings, Jordan; Liu, Qinfang; Lee, Jinhwa; Ma, Wenjun; Blecha, Frank; Miller, Laura C.; Sang, Yongming

Based on sequence analyses, we designed subtype-common or gene-specific primers for expression analyses using quantitative RT-PCR and cloning of coding regions from cDNA pools (Supplementary Material for primer sequence) (9, 13) . For validation of the expression of various porcine IFNs, we amplified cDNA covering whole coding ORFs of representative genes in each gene or subgroup, cloned them in a pcDNA3.3 Topo-mammalian expression vector (Invitrogen, Carlsbad, CA), and confirmed them by sequencing. The cDNA was reverse transcribed from total RNA pools extracted from different tissues with a SuperScript III first-strand synthesis system and random primers (Invitrogen). Coding regions of IFNs were amplified from this cDNA pool for transcription confirmation and building expression constructs. Classification of porcine IFN stimulated genes (ISGs) into tunable or robust subgroups was referred to human or mouse ISGs, and gene-specific primers were designed and validated using porcine gene annotation (Supplemental Excel Sheet). PCR optimization, and real-time RT-PCR analysis were performed as described (9, 10, 13) . In brief, gene-specific or subtype-common primers were designed based on multiple alignments of related IFN sequences, and PCR conditions were optimized and validated using confirmed IFN plasmids to show specific amplification only with templates containing confirmed IFN clone(s). RNA was extracted from tissues and cells as described above. Realtime RT-PCR assays were conducted in a 96-well microplate format using a StepOnePlus TM Real-Time PCR System (Applied Biosystems, Grand Island, NY) with the validated primers. Reactions were conducted with a SYBR Green RT-PCR system (Qiagen, Valencia, CA) with 100 ng of total RNA in a 20µl reaction mixture. Specific optic detection was set at 78 • C for 15 s after each amplification cycle of 95 • C for 15 s, 56-59 • C for 30 s, and 72 • C for 40 s. Critical threshold (Ct) values and melt curves were monitored and collected with the real-time PCR system. Relative gene expression was first normalized against Ct values of the housekeeping gene (βactin) for relative expression levels, and compared with the expression levels of control samples for stimulated regulation (9, 10, 13).