Real time pcr instrument

According to the manufacturer’s method, the liver was homogenized with TRIzol reagent (Gibco-BRL, Rockville, MD, USA), and the total RNA was separated from the aqueous part after centrifugation. Its equal amount in each sample was mixed with Superscript III reverse transcriptase, and a polymerase chain reaction (PCR) was implemented to synthesize cDNA. The synthesized cDNAs were equally added to the SYBR Green mix (Bio-Rad, Richmond, CA) along with the primers for specific genes, and a real-time PCR was conducted in the real-time PCR instrument (Bio-Rad) under optimal conditions for thermal cycling. The expression of the inflammatory cytokine, TNF-α, and interleukin (IL)-1β, were determined using corresponding primers, as previously described [23 (link)]. The expression of the genes of interest was normalized to that of the β-actin gene. A cycle of threshold (CT) for each sample was used for quantifying the expression levels of the interested genes by the comparative CT method (ΔΔCT method). ΔCT was calculated as: ΔCT = CT (target gene) − CT (endogenous reference gene, β-actin). The differences in gene expression were calculated as 2^−ΔΔCT in ΔΔCT = ΔCT for the treatment group − ΔCT for the control group.

The levels of miR-126 in the EPCs, EPC-EXs and astrocytes were determined by Real-time PCR as we previously published [2 (link), 23 (link)]. Briefly, the miRNA was extracted from the EPCs and EPC-EXs by using the TRIzol reagent and the RNA concentration was measured using NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using PrimeScript RT reagent kit (Takara Bio Inc.) following the manufacturer’s instructions. qRT-PCR was carried out using miR-126 specific primers and SYBR Premix Ex Taq kit (Takara Bio Inc.) on a real-time PCR instrument (Bio-Rad). RNA U6 was used as an internal control. The RT primer: 5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GATACG AC CGC ATT-3′. The primers of miR-126: 5′-AGG CGC TCG TAC CGT GAG TAA TA-3′ (forward); 5′-CCA GTG CAG GGT CCG AGG TA-3′ (reverse). The expression of U6 was used as an endogenous control for each sample. The primers of U6: 5′-CTCGCTTCGGCAGCACA-3′ (forward); 5′-AACGCTTCACGAATTTGCGT-3′ (reverse). The expression of miR-126 was normalized to U6 and calculated using 2 − ΔΔCT method.

The sequences of TTCC isoforms, Pgp, and β-actin mRNAs were acquired from the National Centre for Biotechnology Information. Primers (sequences listed in Table 1) were synthesized using Primer-Basic Local Alignment Search Tool online software and ordered from Imperial Life Sciences Pvt. Ltd. (Gurugram, Haryana, India).
Cells were grown in duplicate in 6-well plates, and total RNA was extracted manually using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Next, 1 μg RNA from each group was used to prepare cDNA using the Reverse Transcriptase kit (Qiagen, Hilden, Germany). Real-time PCR was performed using SYBR Green Master Mix (Bio-Rad Laboratories Inc., Hercules, CA) in a real-time PCR instrument (Bio-Rad Laboratories Inc., Hercules, CA). Thermal cycling conditions were as follows: initial denaturation at 95 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 10 s, annealing at 55 °C for 30 s, and extension at 68 °C for 1 min/kb. The final extension was at 68 °C for 10 min. The negative control of each gene was included in the reaction mixture by omitting cDNA samples. mRNA expressions were normalized with β-actin expression. The relative expression of each test gene was measured against the Ca_v3.1 mRNA expression of the negative control group.

The total RNA in liver samples was extracted with Trizol (15596-026, Invitrogen), and the purity and concentration of RNA were detected with nucleic acid protein quantizer (ds-11, Denovix, Wilmington, Delaware, USA). Reverse transcriptase kits (CW2582M, CWBIO, Beijing, China) were used for reverse transcription of RNA into cDNA in strict accordance with the instructions. After primers, cDNA, and SYBR Green Master Mix were mixed thoroughly, and a real-time PCR instrument (Bio-Rad, Hercules, California, USA) was used to amplify and detect fluorescence intensity with the following parameters: 95 °C for 15 min for 1 cycle; 95 °C for 10 s, 53~58 °C for 30 s, and 72 °C for 30 s for 40 cycles. Gene expression was calculated by the 2^−ΔΔCT method, using β-actin as a reference gene. The design of gene primers used for experiments was referred to the primer bank database. In addition, the primer sequences are shown in ds-11-01227-t002">Table 2. Sequences of primers in RT-qPCR.

The total RNA in the liver was isolated using the RNAprep pure tissue kit (Tiangen biotech Co., Ltd., Beijing, China) according to the manutacturer's instructions. The RNA purity was determined by measuring OD₂₆₀/OD₂₈₀ ratio (between 1.8 and 2.1). Reverse transcription was performed using the FastKing gDNA Dispelling RT SuperMix (Tiangen biotech Co., Ltd., Beijing, China) using the following conditions: 42°C for 15 min followed by 95°C for 3 min. Quantitative real-time PCR (qPCR) was performed using the Talent qPCR PreMix (SYBR Green) (Tiangen Biotech Co., Ltd., Beijing, China) under the following conditions: 95°C for 5 min followed by 40 cycles at 95°C for 5 s, 50–60°C for 10 s, and 72°C for 15 s. The CT values for the target genes and β-actin gene (as a housekeeping gene) were calculated by the real-time PCR instrument (Bio-Rad, USA), and the relative gene expression of four genes involved in lipid metabolism, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), HMG-CoA reductase (HMGCR) and cholesterol 7 alpha-hydroxylase (CYP7A1) was calculated using the comparative method 2^−ΔΔCT (26 (link)). The primers sequences used for qRT-PCR in this study were obtained from ThermoFisher (Beijing, China) and shown in Table 1.