Tri rna reagent

Cell culture medium and supplements, such as minimum essential medium (MEM), Roswell Park Memorial Institute medium 1640 (RPMI-1640), neurobasal medium, glutamine, penicillin, streptomycin, fungizone, and trypsin, were acquired from Invitrogen (Waltham, MA, USA). B-27 and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and HyClone (Logan, UT, USA), respectively. The IL-6 ELISA kit was purchased from Abcam (Cambridge, MA, USA). The Griess reagent and CellTiter-Glo^® Luminescence assay kit were purchased from Promega (Madison, WI, USA). Tri-RNA Reagent was purchased from Favorgen (Kaohsiung, Taiwan). The 2× qPCRBIO SyGreen 1step Lo-ROX was obtained from PCR Biosystems (Wayne, PA, USA). 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2HTetrazolium-5-Carboxanilide (XTT), phenazine methosulfate (PMS), Aβ₄₂ peptides, hexafluoropropanol, poly-l-lysine (PLL), dichlorofluorescein diacetate (DCFDA), were obtained from Sigma-Aldrich (St. Louis, MO, USA). Unless otherwise noted, all additional reagents and standards were from Sigma-Aldrich.

Tri-RNA reagent (Favorgen, Pingtung, China) was used to extract total RNA from the cells. Total RNA (1 µg) was used for cDNA synthesis using reverse transcription 5X master mix. Specific primers and iQ™ SYBR green supermix (Bio-Rad Inc., Hercules, CA, USA) were used for the quantification of mRNA expression through a CFX Connect™ (Bio-Rad Inc.). Finally, the 2^−∆∆Ct method was used to evaluate the mRNA levels of individual genes by normalizing to β-actin. The following primers were used: MMP2 (rat), forward-5′-GATACCCCAAGCCACTG-3′ and reverse-5′-TCCAAACTTCACGCTCTT-3′; MMP9 (rat), forward-5′-CAGACCAAGGGTACAGCCTGTT-3′ and reverse-5′-AGCGCATGGCCGAACTC-3′; α-SMA (rat), forward-5′-CATCACCAACTGGGACGACA-3′ and reverse-5′-TCCGTTAGCAAGGTCGGATG-3′; OPN (rat), forward-5′-GCT CTCAAGGTCATCCCAGTTG-3′ and reverse-5′-TGTTTCCACGCTTGGTTCACT-3′; vimentin (rat), forward-5′-AGGGGAGGAGAGCAGGATTT-3′ and reverse-5′-GGAGTGGGTGTCAACCAGAG-3′; β-actin (rat), forward-5′-ATGGATGACGATATCGCTGCG-3′ and reverse-5′-CAGGGTCAGGATGCCTCTCTT-3′.

qPCR was performed as previously described [13 (link)]. Total RNA was isolated using Tri-RNA reagent (FAVORGEN, Ping-Tung, Taiwan), and 1 μg of total RNA was used to synthesize cDNA using RNA-to-cDNA EcoDryTM premix (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol. Relative mRNA levels were evaluated in ViiA7 (Applied Biosystems, Foster City, CA, USA) using the SYBR Green PCR Master Mix (Applied Biosystems). Target gene expression was normalized to that of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences used in this study are listed in Table 1.

Total RNA of ASCs was extracted using Tri-RNA reagent (Favorgen, Ping-Tung, Taiwan) according to the manufacturer’s instructions. Reverse transcription was performed with 1 μg of RNA, random primers, and M-MLV Reverse transcriptase (Promega, MI, USA). The primers used for SYBR Green reverse-transcription qPCR (RT-qPCR) were as follows: IL-6, forward 5′-CACACAGACAGCCACTCACC-3′ and reverse 5′-TTTTCTGCCAGTGCCTCTTT-3′; vascular endothelial growth factor (VEGF), forward 5′-TCTTCAAGCCATCCTGTGTG-3′ and reverse 5′-ATCTGCATGGTGATGTTGGA-3′; hepatocyte growth factor (HGF), forward 5′-TGCTGTCCTGGATGATTTTG-3′ and reverse 5′-AGTGTAGCCCCAGCCATAAA-3′; NAD-dependent deacetylase sirtuin-1 (SIRT1) [16 (link)], forward 5′-AGAACCACCAAAGCGGAAA-3′ and reverse 5′-TCCCACAGGAGACAGAAACC-3′; and GAPDH, forward 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′. RT-qPCR was performed with the Applied Biosystems 7500 Fast Real-Time PCR System (Life technologies, Carlsbad, CA, USA). After normalizing to the GAPDH gene, expression levels for each target gene were calculated using the comparative threshold cycle (CT) method. Data are presented as the mean ± standard deviation (SD) from three independent experiments.

Total RNA was isolated from cells using Tri-RNA Reagent (FAVORGEN). After cDNA synthesis, the cDNA was quantified and then subjected to analysis of mRNA expression. The PCR primers used are presented in Supplementary Table S1. Dissociation curves were examined after each PCR run to ensure amplification of a single product of the appropriate length. The mean threshold cycle (C_T) and standard error values were calculated from individual C_T values obtained from triplicate reactions per stage. The normalised mean C_T value was estimated as ΔC_T by subtracting the mean C_T of β-actin. The value ΔΔC_T was calculated as the difference between the control ΔC_T and the values obtained for each sample. The n-fold change in gene expression, relative to an untreated control, was calculated as 2^−ΔΔCT.