Ncode express sybr greener mirna qpcr kit

Total RNA was isolated from BMSCs of OVX and control using miRcute miRNA isolation kit (TIANGEN, China, DP501), and 1 μg RNA from each sample was reverse-transcribed into cDNA using the NCodeTM EXPRESS SYBR GreenERTM miRNA qPCR kit (Invitrogen). The qPCR reaction was performed using the GeneAmp PCR system 9600 (Perkin Elmer). All specific primers are listed in Table 4, and were synthesized by Sangon Biotech (Sangon Biotech, Shanghai, China). Relative transcript levels of circRNAs and mRNAs were normalized with β-actin, while those of miRNAs were normalized with U6. The expression level of each mRNA, miRNA, and circRNA was calculated using comparative Ct method.Table 4

The probe sequences and primers for qRT-PCR in the experiment.

Gene	Primer sequence
circRNA_3832	F: CAATGACACTGGGACAGACG
	R: GTTTGGGGTGAGTGTTTGCT
circRNA_0020	F: CCTGAGAGATTTTAGTTCGAGGT
	R: TTTGAACTGCGAGACACTGG
Runx3	F: CAGGTTCAACGACCTTCGATT
	R: GTGGTAGGTAGCCACTTGGG
Nnmt	F: AGCACAAGACGTGAGCGTAA
	R: CGGATATCCAAAGGGGGCTC
Col3a1	F: AAGGCTGCAAGATGGATGCT
	R: GTGCTTACGTGGGACAGTCA

Seven DElncRNAs were validated by qPCR. The cDNAs were reversed transcribed from RNA utilizing the NCode^TM EXPRESS SYBR^® GreenER^TM miRNA qPCR kit (Invitrogen, Carlsbad, CA, United States). We conducted qPCR reaction on the GeneAmp PCR system 9600 (Perkin Elmer, United States). All primers were obtained from Sangon Biotech (Shanghai, China), and their sequences are shown in Table 1. The relative lncRNAs expression was obtained after normalization to β-actin. 2^–ΔΔCt method was applied to calculate the expression level of each lncRNA.