Sybr green real time pcr master mix

Liver samples were harvested from mice and homogenized. Total RNA was isolated using RNeasy kit (Qiagen, Valencia, CA) and reverse transcribed by iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. Quantitative Real time PCR was performed on a BioRad Cycler (Biorad, Hercules, CA, USA) by using SYBRGreen real time PCR master mix (Kapabiosystem, Woburn, MA, USA) according to the manufacturer’s instructions. The housekeeping gene 18S was used as an internal control. The PCR primers used to amplify each gene are listed in Table S2. For isolation and quantification of miR-122 and miR-192, platelet-free plasma was isolated from CDAA-fed, high fat or control mice and incubated with 10 µg/mL of RNase (Roche, Indianapolis, IN, USA) for 30 min at 37°C to remove any RNAs adhering to the external leaflet of circulating EVs. Circulating EVs were then ultracentrifuged at 100,000 g for 90 min at 10°C. Total encapsulated RNAs in EVs or in liver specimens, including miRNAs, were isolated by miRNeasy Mini kit (QIAGEN). CDNA was synthesized using specific miRNA primers (Applied Biosystems) in TaqMan microRNA Reverse Transcription kit (Applied Biosystems). MiRNA abundance was detected with TaqMan probe (Applied Biosystems) on 7300 Real time PCR system (Applied Biosystems). The U6 snRNA was used as an internal control and to normalize miR-122 and miR-192 expression.

Total RNA was purified from all T-OPLL patient blood using the SK1321 RNA Blood Mini Kit (Sangon Biotech Co., Ltd., Shanghai, China). A one-column DNase digest (Sangon Biotech Co., Ltd.) was performed before the clean-up step to eliminate residual genomic DNA. cDNA was synthesized from total RNA (2 μg) using a RevertAid Premium Reverse Transcriptase kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Relative qPCR was applied to quantify the mRNAs levels of COL6A1 using SYBR Green Real-Time PCR master mix on the LightCycler480 Real-Time System (Roche Diagnostics, Basel, Switzerland). All experiments were performed in triplicate and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Details of the primer sequences are listed in Table 1.
Table 1

Primer sequences used for quantitative polymerase chain reaction

Gene	Primer sequence
COL6A1	Forward 5′-CGAGATTGCCAAGGACTTCG-3′ Reverse 5′-AGGCTCTTGATGGCTTCCTT-3′
GAPDH	Forward 5′-TGGGTGTGAACCATGAGAAGT-3′ Reverse 5′-GAGTCCTTCCACGATACCAA-3′

RNA of iPSCs and FLS cells was extracted using Trizol (Invitrogen). We synthesized cDNA from RNA through RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific). We performed reverse transcriptase (RT) polymerase chain reactions (PCRs) using the synthesized cDNA synthesized from iTaq DNA Polymerase Kit (iNtRON Biotechnology). Quantitative real-time PCR was performed with the LightCycler® 480 instrument (Roche), the SYBR Green Real-time PCR Master Mix (Roche). Gene expression levels were normalized to GAPDH expression levels. The primer sequences are presented in Additional file 5: Table S1.

Total RNA (1 μg) was reverse‐transcribed using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biomedical Technology) Quantitative PCR was then used to confirm the significantly altered genes revealed in the RNA‐Seq analyses. The qRT‐PCR primers to determine the target gene expression levels were shown in Table S1. Each quantitative real‐time PCR was carried out in triplicate with SYBR Green Real‐time PCR Master Mix (Roche). The expression levels were normalized to the 18S in each sample.

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, California) and then used to synthesize cDNA using the FastQuant RT Kit (KR106; TIANGEN, Beijing, China). Sequences of the primers used for the PCR analysis are listed in Table S1. Quantitative PCR was performed according to a standard protocol using the SYBR Green Real-Time PCR MasterMix (Roche, Basel, Switzerland) in a Roche LightCycler 96 System. The PCR conditions were: 94 °C for 180 s, followed by 50 cycles of 94 °C for 10 s, 65 °C for 15 s, and 72 °C for 15 s; followed by 95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s. Relative mRNA expression levels of the gene of interest were calculated using the 2^-△△Ct method.