Rnase inhibitor

Total RNA was isolated from mesenchymal and myoblast-like cells using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer's recommendations. Complementary DNA (cDNA) was prepared from each total 5 μg RNA sample by incubating a 25 μL mixture containing 0.25 mol·L⁻¹ dithiothreitol, 5 × reaction buffer, RNase inhibitor, and avian myeloblastosis virus reverse transcriptase (Takara Bio, Shiga, Japan) for 1 h at 41 °C. This cDNA was used as a template for PCR amplification. Amplifications (0.5 μL cDNA in a total reaction volume of 50 μL) were performed at 95 °C for 1 s, at 52 °C for 30 s, and at 72 °C for 20 s (3 cycles); followed by 95 °C for 30 s, at 52 °C for 30 s, and at 72 °C 20 s (25 cycles) using a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). Primer sequences, reaction conditions, and the size of each product are listed in Table 1. The amplification of glyceraldehyde-3-phosphate dehydrogenase was performed in the same manner to evaluate the cDNA quality. Amplification products were separated by electrophoresis on 1.5% or 2.0% agarose gels.

Cells were treated with 100 μg/ml CHX for 10 min prior to being harvested. Cells washed with ice-cold PBS containing 100 μg/ml CHX were lysed in 550 μl of polysome lysis buffer (50 mM Tris-HCl at pH 7.5, 100 mM NaCl, 30 mM MgCl₂, and 0.1% NP-40) containing a protease inhibitor cocktail, 100 μg/ml CHX and 40 U/ml RNase inhibitor (Takara Bio, Shiga, Japan) at 4 °C for 30 min. The lysate was centrifuged for 10 min at 13,000 ×g. The supernatant (500 μl) was loaded onto a 10–50% sucrose gradient in polysome lysis buffer. The gradient was subjected to centrifugation in a Beckman SW55Ti rotor at 100,000 xg at 4 °C for 1 h, following which fifty 100-μl fractions were collected from the top. Absorbance was measured at 260 nm for each fraction.

To evaluate the osteogenic gene expression in ADMPC, 1 × 10⁵ cells were cultured for 3 weeks with or without ODM. Total RNA from cultured cells was isolated using ISOGEN II (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. cDNA was synthesized from 1 µg of total RNA in a 20 µL reaction containing 10X reaction buffer, 1 mmol/L of dinitrophenol phosphate (dNTP) mixture, 1 U/ L RNase inhibitor, 0.25 U/ L reverse transcriptase, and 0.125 mol/L random hexamers (Takara, Tokyo, Japan). The cDNA was then amplified, and specific gene expression using swine-specific primers (Supplementary Table S2, Osteogenic genes) was quantified using a real-time PCR apparatus (Bio-Rad CFX Connect, Applied Biosystems) for 40 cycles following the reaction profile: 95 °C for 3 mins, 55 °C for 30 sec, 65 °C for 5 sec. The expression of the tested osteogenic genes was calculated using the 2^−ΔCT method compared with housekeeping gene GAPDH.

Cerebral cortices and hippocampi from 34-week old wild-type and tau-knockout CB57BL/6J mice were prepared by the Institute of Immunology, Utsunomiya (Japan). Briefly, brains were washed with ice-cold PBS and homogenized in TKM buffer (50 mM triethanolamine [pH 7.8], 50 mM KCl, 5 mM MgCl₂, 0.25 M sucrose, 1 mM PMSF, protein inhibitor [complete cocktail without EDTA, Roche], 1 mM DTT and RNase inhibitor [0.2 unit/μl, Takara]). Homogenates were centrifuged (1000 × g, 10 min, 4 °C) and 0.5 ml of each supernatant was loaded onto a 15–45% sucrose gradient (9 ml) with a 0.75 ml cushion (45% sucrose) before centrifugation at 36,000 rpm (2 h, 4 °C, Beckman Coulter SW40Ti rotor). The gradient was fractionated, and the distribution of RNAs and ribosomal protein in each fraction was monitored by absorbance at 254 nm and Western blotting of S6 ribosome protein, respectively.

Total RNA was extracted using the acid guanidium thiocyante/phenochloroform method, using a total RNA isolation reagent (Trizol Reagent, Invitrogen,USA) according to the manufacturer’s instructions. The RNAs were reverse-transcribed to cDNAs in a Thermal Cycler using Oligo (dT) primer (Invtirogen), RNase Inhibitor (Takara), Reverse transcriptase (Takara), dNTP Mixture and RNA PCR buffer (Sigma). A quantitative RT-PCR assay was carried out with a LightCycler™ (Roche Diagnostics, Mannheim, Germany) using the double-stranded DNA dye SYBR Green 1 (Roche Diagnostics) in order to observe the level of mRNAs. Primer sequence and size used in this study were shown in Table 2. Quantification was performed by comparing the levels obtained with standard samples as a previous study [21 (link)]. In the present study, the concentrations of cDNA in the unstimulated samples were 0.2, 0.5, 1.0 and 2.0 μl. Melting curve analyses were performed after the PCR amplification and to confirm no primer dimmer in the PCR products. The ratios of HSP70 mRNA expression were adjusted by the value of the housekeeping gene GAPDH.Table 2

Primer sequence and size used in this study

Gene	Sequence		Size
HSP70	Forward	5’-CGGACGAGTACAAGGTTGA-3’	206
	Reverse	5’- CTCTTTCTCCGCCAACTG-3’
GAPDH	Forward	5’-AGGGGCTCTCCAGAACATCA-3’	196
	Reverse	5’-GCCTGCTTCACCACCTTCTT-3’

The viral RNA in the supernatants was extracted with a viral RNA minikit (catalog no. W7091; Watson Biotechnologies). Synthesis of cDNA was performed in a 20-μl volume containing 200 ng of total RNA, 20 U of avian myeloblastosis virus (AMV) reverse transcriptase (catalog no. D2620; TaKaRa), 200 μM deoxynucleoside triphosphates (dNTPs) (catalog no. D4030A; TaKaRa), 0.4 μM random primers (catalog no. D6045; TaKaRa), 0.5 μl of RNase inhibitor (catalog no. D2313A; TaKaRa), and 4 μl of 5× AMV reverse transcriptase buffer. Quantification of CSFV genome copy numbers was performed with Premix Ex Taq (Probe qPCR) (catalog no. RR390A; TaKaRa) using a previously described real-time reverse transcription-PCR (RT-qPCR) assay (34 (link)).