Isogen

Total RNA was isolated from DPSCs cultured in NM or OM with or without TH using ISOGEN (Invitrogen, Carlsbad, MA, USA). Reverse transcription was performed using a QuantiTect Reverse Transcription kit (Qiagen, Venlo, Netherland) in accordance with the manufacturer’s instructions. Real-time polymerase chain reaction was performed in a Light Cycler 96 (Roche Diagnostics, Basel, Switizerland) using THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) under the following conditions: 95 °C for 60 s and then 45 cycles at 95 °C for 10 s, 65 °C for 30 s, and 72 °C for 45 s. Real-time polymerase chain reaction was conducted to analyze ALP and COLIA1, early markers of osteoblast differentiation, osteocalcin, a mature osteoblast marker, Runx2, an osteoprogenitor marker, and COX2, a marker enzyme regulating prostaglandins. GAPDH was used as an endogenous control. All reactions were run in triplicate. The primer sequences are presented in Table 1.

Total RNA was extracted from the spleens of the vaccinated pigs using ISOGEN (Invitrogen). Reverse transcriptase-PCR (RT-PCR) was monitored using a one-step RNA PCR kit (Takara) with three pairs of primers according to the operating instructions. Primers are listed below: IFN-Υ (5′-GTTTTTCTGGCTCTTACTGC-3′; 5′-CTTCCGCTT TCTTAGGTTAG-3′) (Chaoprasid and Dersch, 2021 (link)); TNF-α (5-ACTGCACTTCGAGGTTATCGG-3′, 5′-GGCGACGGGCTTATCTGA-3′) (Meissonnier et al., 2008 (link)); interleukin (IL)-4 (5′-GTCTGCTTACTGGCATGTACCA-3′; 5′-GCTCCATGCACGAGTTCTTTCT-3′) (Duvigneau et al., 2005 (link)); GAPDH (5′-AACGACCCCTTCATTGAC-3′; 5′-TCCACGACATACTCAGCAC-3′).
Quantitative reverse transcriptase-PCR (qRT-PCR) was performed on the 7900HT Sequence Detection System (Applied Biosystems) using SYBR Green (Meissonnier et al., 2008 (link)). Each sample was analyzed in triplicate. Data were analyzed by a comparative CT method (Applied Biosystems). Transcript levels were calculated by normalizing to the levels of GAPDH mRNA. In addition, cytokines (IFN-γ, IL 4, TNF-α) in the spleen were analyzed in relation to the expression of antibodies (IgG, IgA) in the mucus, MLN and serum utilizing JMP software.¹

To analyze gene expression, total RNA was isolated from DPSCs that were cultured in differentiation medium using ISOGEN (Invitrogen, Carlsbad, MA, USA). Reverse transcription was performed with a QuantiTect Reverse Transcription kit (Qiagen, Venlo, The Netherlands) in accordance with the manufacturer’s instructions. Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed on a Light Cycler 96 (Roche Diagnostics, Basel, Switzerland) using THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, Osaka, Japan). The RT-PCR conditions were as follows: 95 °C for 60 s, then 45 cycles of 95 °C for 10 s, 65 °C for 30 s, and 72 °C for 45 s. GAPDH served as the internal control, and each reaction was replicated three times. Differences in the relative expression among samples were calculated by the ΔΔCT method. All primer sequences are listed in Table 1.