Rneasy rna isolation minikit

Following the manufacturer’s instructions, total RNA was further purified using a Qiagen RNeasy RNA isolation minikit (catalog number 74,104, Germantown, MD, USA). By using the ProtoScript II First Strand cDNA synthesis kit and 1 µg of total RNA per reaction, complementary DNA (cDNA) was synthesized (New England Biolab, cat# E6560S, Ipswich, Massachusetts, USA). iQ SYBR green supermix (Biorad, cat# 1708880, Des Plaines, IL, USA) and Bio-Rad CFX96 Fast Real-Time PCR Systems were used to perform qPCR.100ng of cDNA was used per RT-PCR reaction. The ΔΔCt relative quantification method was used to determine changes in gene expression. Actin mRNA was used as an internal control. Each value is represented by its mean ± standard error (SEM).

RNAs were extracted from BA-MSCs by using the RNeasy RNA Isolation Mini Kit (#74104, Qiagen). Reverse transcription was performed using the Sensiscript RT kit (#205211, Qiagen) according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) reactions were performed with the Tianlong Real-Time PCR System by using the Fast Essential DNA Green Master Kit (#06402712001, Roche). The qRT-PCR conditions were as follows: at 95°C for 10 min; and then 40 cycles, including one step at 95°C for 20 seconds, one step at 55°C for 20 seconds, and 72°C for 20 seconds; and the last cycle at 95°C for 10 seconds, 65°C for 1 min, and 97°C for 1 second. Relative expression of genes was determined by cycle threshold (Ct) value and normalized to the internal control GAPDH. The primer sequences used in this study are listed in Table S1.

Total RNA was isolated using a RNeasy RNA isolation minikit (Qiagen, cat# 74104, Germantown, MD, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using 1μg of RNA/reaction using ProtoScript First Strand RNA synthesis reverse transcriptase and oligo (dT) primers (New England Biolab, cat# E6300L, Ipswich, MA, USA). qPCR was conducted using iQ SYBR green supermix (Biorad, cat# 1708880, Des Plaines, IL, USA) and Bio-Rad CFX96 Fast Real-Time PCR Systems. Gene expression changes were determined by ΔΔCt relative quantification method. GAPDH mRNA was used as an internal control. All values are presented as mean ± standard error mean (SEM).