Hybrid rtm

Viral RNA was extracted from 100 µL of serum and 1g of lung samples using a viral RNA extraction kit (MagMAX™ Viral RNA Isolation Kit, Life Technologies) and a total RNA extraction kit (Hybrid-RTM, GeneAll, Seoul, Korea) according to the manufacturer’s instructions. The virus levels in serum and lungs were measured using a real-time reverse transcription-polymerase chain reaction (RT-PCR) employing a one-step reverse transcriptase kit (AgPath-IDTM One-Step RT-PCR Kit, Ambion, Austin, TX, USA) with the 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Primers and aminor groove binder (MGB) fluorescent probe specific to a conserved region of ORF7 were used as described previously [24 (link)].

RNA acquisition, cDNA synthesis, and RT-PCR were performed using methods described by Ko et al. [28 (link),29 (link)]. The relative mRNA expression of each target gene was normalized to GAPDH [30 (link)]. The total RNA was evidence-based complementary and alternative-medicine-isolated from each cell treatment using Hybrid-R^TM (GeneAll, Seoul, Republic of Korea) [31 (link),32 (link)]. Total RNA (500 ng) was converted to cDNA using oligo (dT) at incubation conditions of 55 °C for 60 min followed by 85 °C for 5 min and then stored at 4 °C until further use. Real-time quantitative PCR was performed using the Universal SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA). cDNA was amplified using the following conditions: 95 °C for 15 min followed by 40 cycles at 95 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s. Real-time PCR analysis was performed on an Applied Biosystems StepOne system (Applied Biosystems, USA) [33 (link)]. In this study, quantification based on the relative expression of a target gene versus GAPDH gene (2^−ΔΔCt) was employed to determine the level of mRNA expression. The primer sequences used for RT-PCR analysis were as follows (Table 1).

ELISA kits (R&D Systems, Abingdon, UK) were utilized to measure the serum levels of TNF-α, interleukin (IL)-1β, IL-6, and IL-10, following the manufacturer’s instructions. In ice-cold RIPA buffer (Invitrogen, Carlsbad, CA, USA) with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA), the colon tissues were homogenized. SDS-PAGE was used to separate the protein samples (25 μg per lane), which were then transferred to polyvinylidene difluoride membranes (Bio-Rad, Munich, Germany). The blots were examined with the designated antibodies and produced using an enhanced chemiluminescence (ECL) system (Amersham, Buckinghamshire, UK). Densitometric scanning (Amersham Imager 600; GE Healthcare, Buckinghamshire, UK) was used for the quantitative analysis. Quantitative real-time reverse transcription PCR (qRT-PCR) was used to measure the mRNA expression levels of inflammatory cytokines. Colon tissues were homogenized using Hybrid-RTM (GeneAll, Seoul, Korea). A cDNA Synthesis Kit (BioFact, Daejeon, Korea) was then used to reverse transcribe 1 μg of RNA in order to create cDNA. The 2× RT Pre-Mix (BioFact) was used to carry out the qRT-PCR. Table 2 provides a list of the used primer sequences. The comparative Ct method was used to determine the relative mRNA levels, with β-actin serving as the reference gene.