Ez probe qpcr master mix for microrna

Total RNAs were isolated from PDLSCs using EZ-press RNA Purification Kit (EZBioscience, USA), mRNAs were reverse-transcribed into cDNAs by the Color Reverse Transcription Kit (EZBioscience, Roseville, USA) and the cDNAs of miRNAs were acquired with the microRNA Reverse Transcription Kit (EZBioscience, Roseville, USA). 2×Color SYBR Green qPCR Master Mix (for mRNA) and EZ-Probe qPCR Master Mix for microRNA (EZBioscience, Roseville, USA) were used for subsequent qRT-PCR amplification on ABI Quant-Studio 5 system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were used as internal controls. The sequences of the gene-specific primers are listed in Table 1. Universal 3'qPCR primer is included in EZ-Probe qPCR Master Mix for microRNA (EZBioscience, Roseville, USA).Table 1

Primer sequences for quantitative reverse-transcription polymerase chain reaction

Gene	Sequence 5′ → 3′
SMAD6	Forward: AGACGGCGTTGGCCTTT
	Reverse: CCTGCCTTTACCTTGCCTTTT
LOC100302640	Forward: GCGGAAGGGGCTTGTTCATT
	Reverse: TGCGTAGGTCAAGTATCGGC
miR-1469	Forward: CTCGGTGCGGGGCG
U6	Forward: CCTGCTTCGGCAGCACA
GAPDH	Forward: AACGGATTTGGTCGTATTGGG
	Reverse: CCTGGAAGATGGTGATGGGAT

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenas; SMAD6, Mothers against decapentaplegic homolog 6.

Total RNA was isolated using TRIzol reagent (Invitrogen, CA, United States). For lncRNAs and mRNAs, the extracted RNA was reverse transcribed into cDNA using Color Reverse Transcription kit (EZBioscience, Roseville, United States), and for miRNAs, the extracted RNA was reverse transcribed into cDNA using microRNA Reverse Transcription kit (EZBioscience, Roseville, United States) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to select mRNAs (Ttr, Tfpi2, Ccl2, Lhx5 and Cxcl1) and lncRNAs (Rph3a, Smim13, AABR07066379.1, LINC2085 and Lcorl) using SYBR Green qPCR Master Mix (EZBioscience, Roseville, United States) and to select miRNAs (miR-450b-3p, miR-200b-3p, miR-200a-3p and miR-429) using EZ-Probe qPCR Master Mix for microRNA (EZBioscience, Roseville, United States) according to the manufacturer’s instructions. Relative expression levels were normalized to GAPDH and U6 as internal standard controls for selected DERNAs, and they were calculated by the 2^−ΔΔCt method. Specific primers were designed as shown in Table 1.