Sybr green fluorophore

Total RNA was extracted with TRIzol reagent. Total RNA concentrations were measured using ND-1000 (NanoDrop Technologies), and cDNA was synthesized with high-capacity cDNA reverse transcription kits (Applied Biosystems, F. Hoffmann-La Roche Ltd., Switzerland), according to the protocol provided by the manufacturer. PCR primers for TGF-β1, Smad2, Smad3, Smad7, and GAPDH were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences of the forward and reverse primers used for amplification are shown in Table 1. Real-time PCR was performed using a one-step real-time PCR system (Applied Biosystems) with SYBR green fluorophore. Each sample for reactions was run in at least duplicates. Threshold cycle (Ct) data were collected by an inherent system and posted on an Excel sheet. GAPDH was performed as an internal control. 2^−ΔΔCt was calculated to evaluate mRNA fold change relative to GAPDH [26 (link)].

The expression of MyoD1a, myogenin and Cadherin 15 (M-cadherin) that exhibited distinct temporal expression pattern as revealed by microarray experiment, was analysed by qPCR using a real-time PCR kit incorporating a SYBR® Green fluorophore (Applied Biosystems). The relative abundance of target cDNA within the sample set was calculated from a serial dilution (1:1–1:256) (standard curve) of pool cDNA using StepOneTM Software V2.0.2 (Applied Biosystems). Subsequently, real-time PCR data were normalised by dividing the raw data by the eF1α gene expression value.

Total RNA was extracted using the Trizol reagent (Invitrogen), and 20 μg of total RNA was used to synthesize first-strand cDNA using 2 μM oligo-dT18 primers with M-MLV reverse transcriptase (Promega, USA) according to the manufacturer’s instructions. Specific primers were designed by PRIMER3plus (software (http://primer3plus.com/cgibin/dev/primer3plus.cgi); qRT-PCR was performed using SYBR Green fluorophore (Applied Biosystems) on a Bio-Rad CFX Real-Time Thermocycler (Hercules, CA, USA) by Qin et al. [30 (link)]. Three biological experiments were repeated, and the threshold cycles (Ct) of each test target were averaged for triplicate PCR reactions. The relative expression ratio of the TdLTP2 gene was calculated by using the comparative CT method with the actin gene (actin_Fw: 5′-TCCCTCAGCACATTCCAGCAGAT-3′ and actin_Rv: 5′- AACGATTCCTGGACCTG CCTCATC-3′) as an internal expression standard [31 (link)]. The relative expression level was calculated from triplicate measurements based on the 2^−DDCT equation: DDCT = (CT_Tstress − CT_UBQ10stress) − (CT_Tcontrol − CT_UBQ10control), where CT_Tstress is the CT of the target gene from stressed samples, CT_UBQ10stress is the CT of the UBQ10 gene in stressed samples, CT_Tcontrol is the CT of the target gene from control samples and CT_UBQ10control is the CT of the UBQ10 gene in control samples.