Nanodrop assay

The mRNA of cells was extracted using trizol (Invitrogen) and collected using the Qiagen RNEasy Extraction kit (Qiagen). Samples were stored at -80°C until reversed transcription. Total RNA concentration and purity were detected by Nanodrop Assay (Tecan M200). The first strand cDNA was synthesized by reverse transcriptase as described of M-MLV manual (Promega). Gene-specific primers including GAPDH, COL I, COL II, OPN, and Runx2 were designed using the primer design software of beacon 5.0 (Table 1). All samples were performed in triplicates in 8 striped optical tube (Axygen) using qPCR SYBR Green Mix Kit (Stratagene) The amplification efficiencies of primers was verified by cDNA serial 5 times dilutions. The qPCR amplification was done as follows: initial heating at 95°C for 10min, followed by 40 cycles at 95°C for 30 s, 58°C for 60s, 72°C for 60 s. Specificity of listed oligonucleotides were checked by BLASTN^® (Basic Local Alignment Search Tool) against the mouse RefSeq RNA database at NCBI.

The MC3T3-E1 cells cultured on various nanofibrous matrices for 7 days were also collected for the evaluation of the osteogenesis-related genes expression. Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. The total RNA concentration and purity were detected by a Nanodrop assay (Tecan M200), and the first strand cDNA was synthesized by reverse transcriptase as described in theM-MLV manual (Promega). The expression of osteogenic markers was quantified by qPCR SYBRGreen Mix Kit (TaKaRa). The primer sequences specific for the target gene including anti-runt-related transcription factor 2(RUNX2), osteopontin (OPN) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used for qRT-PCR are listed in Table 1. The specificities of the listed oligonucleotides were checked by BLASTN® (Basic Local Alignment Search Tool) against the mouse RefSeq RNA database at NCBI. The qPCR amplification was done as follows: initial denaturation at 95°C for 10min, followed by 40 cycles at 95°C for 30 s, 58°C for 1 min, 72°C for 1 min. The comparative threshold cycle method was used to analyse the Q-PCR results using iCycleriQ Detection System software with GAPDH as the reference gene. All results were quantified using the ΔΔCt relative quantification method.