Thermoscript reverse transcription pcr system

Total RNA was extracted from VEGF treated and untreated MSCs using TRIZOL (SIGMA, USA) and reverse transcribed into complementary DNA (cDNA) with the ThermoScript reverse transcription-PCR system (Invitrogen, CA, USA). The qRT-PCR was performed by using SYBR green kits (Applied Biosystems, The Netherlands). Results were analyzed after 45 cycles of amplification using the ABI 7500 Fast Real Time PCR System. Primers were designed on the basis of the reported sequences (Primer bank NCBI; CTGF: 5′-CAGCATGGACGTTCGTCTG-3′ (forward) and 5′-AACCACGGTTTGGTCCTTGG-3′ (reverse); Cav-1: 5′-AATACTGGTTTTACCGCTTGCT-3′ (forward) and 5′-CATGGTACAACTGCCCAGATG-3′ (reverse); β-actin: 5′-CCTGGCACCCAGCACAAT-3′ (forward) and 5′-AGTACTCCGTGTGGATCGGC-3′ (reverse)). The qRT-PCR was run in triplicate.

Primary rat chondrocytes were treated with 50 or 100 μM acteoside in the presence or absence of 10 ng/mL IL-1β for 24 h. Thereafter, total RNA was isolated from the primary rat chondrocytes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA concentration was measured using a NanoDrop 2000 (Thermo Scientific, Rockford, IL, USA). To synthesize cDNA, 1 μg RNA was reverse transcribed using a ThermoScript reverse transcription-PCR system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. qPCR of cDNA was performed using 2× TOPsimple™ DyeMIX-nTaq (Enzynomics, Seoul, Republic of Korea) and specific primers on a TaKaRa PCR Thermal Cycler Device (TaKaRa Bio Inc., Shiga, Japan). Thereafter, the PCR products were electrophoresed on an agarose gel to determine the expression levels of target genes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. In addition, for qRT-PCR, cDNA was amplified using an Eco™ Real-Time PCR system (illumine Inc., San Diego, CA, USA). β-Actin was used as an endogenous control. The sequences of the primers used in the qPCR and qRT-PCR are summarized in Tables 1 and 2, respectively.

Total RNA was extracted from 20 mg of liver tissue using TRIzol reagent (Invitrogen, Zug, CH) and transcribed into cDNA using the ThermoScript reverse‐transcription PCR System (Invitrogen). TaqMan gene expression assays for Ccnd1 (Mm00432359_m1), Ccne1 (Mm00432367_m1), Ccna2 (Mm00438064_m1), Ccnb2 (Mm01171453_m1), Yap1 (Mm01143263_m1), Ctgf (Mm01192932_g1), Birc5 (Mm00599749_m1), Myc (Mm00487803_m1), Cyr61 (Mm00487498_m1), and 18S rRNA internal control (TaqMan ribosomal RNA control reagents) were from PE Applied Biosystems (Rotkreuz, CH). The results represent fold induction (2^−∆Ct) of mRNA expression ±SD.

Total RNA was obtained from cultured primary human AECII, MRC5, and sorted epithelial cells using RNeasy kit (Qiagen, 74,004) following the manufacturers’ instructions. extracted and followed by reverse transcription into complementary DNA with the ThermoScript reverse transcription PCR system (Invitrogen, CA, USA) for the subsequent qPCR analysis. qPCR was performed using SYBR Green system (ThermoFisher), and results were analyzed after 40 cycles of amplification using ABI 7500 fast real-time PCR system. Relative expression levels of genes were defined by ∆∆Ct method and normalizing to β-actin. The primer sequences were listed in Additional file 1: Table S3.