7300 fast real time pcr system

After the transfection of the cells with the miR-486 mimics/inhibitors, total RNA was purified using Trizol reagent (Invitrogen) following the manufacturer’s instructions. cDNA (complementary DNA) was synthesized using the PrimerScript RT-PCR kit (TaKaRa) according to the manufacturer’s instructions.
The expression of miR-486 and the 5S internal control gene were quantified using real-time PCR quantification and the Hairpin-it miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, China) according to the manufacturer’s protocol. Specific primers for miR-486 and 5S were designed by GenePharma. The expression of these genes was analyzed using a 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
The expression of PTEN, AKT, mTOR, and β-actin was quantified using Real-Time-PCR quantification and SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer’s instructions. β-actin was used as a loading control. The primers that were used for qRT-PCR are listed in Table 1. All qPCR reactions were performed in triplicate.

A piece of renal cortical tissue was stored in RNA-later (Sigma) overnight at 4 °C. We used 7300 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) to measure 18S, transforming growth factor (TGF)-β, and tumour necrosis factor (TNF)-α mRNA levels. The mouse primer sequences (forward and reverse) were as follow: 18S: 5′-GTAACCCGTTGAACCCCATT-3′, 5′-CCATCCAATCGGTAGTAGCG-3′; TGF-β: 5′-GCTGCTGACCCCCACTGATA-3′, 5′- ACAAGAGCAGTGAGCGCTGAA-3′; and TNF-α: 5′-AGCCTGTAGCCACGTCGTA-3′, 5′-TGGCACCACTAGTTGGTTGTCT-3′. Relative mRNA levels were determined using the 2^−ΔΔCt method. The ddCt value was calculated using data from the normal control group.

The detailed method and DNA primer sequences were described previously [7 (link)]. RAW264.7 murine macrophage-like cells and CT26.WT murine colorectal cancer cells (CRL-2638) were purchased from RIKEN BRC (Tsukuba, Japan) and American Type Cell Collection (Manassas, VA, USA), respectively. CT26 cells (4 × 10⁶) were grown in T75 flask with 20 mL of RPMI medium containing 10% FBS for 3 days. The culture medium (CT26-CM) was collected and centrifuged at 400 g for 5 min. RAW264.7 cells (2.4 × 10⁵) were incubated in 24-well plate in the presence or absence of KS-133 (KS-133 concentration = 1, 3, or 10 μM, DMSO concentration = 0.1%) with 20% CT26-CM/DMEM for 3 days. During the incubation, medium containing KS-133 was replaced every day. mRNA extraction was performed using ISOGEN (Nippon Gene, Tokyo, Japan) on day 3 after CT26-CM incubation, and cDNA was prepared. The gene expression of M1 markers (TNF-α, iNOS, CXCL10), M2 markers (Mrc-1, IL-1rn, CCL22), and β-actin was analyzed by real-time polymerase chain reaction (PCR) using a 7300 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, MA, USA,) and Light Cycler Fast Start DNA Master kit (Applied Biosystems). The relative mRNA expression was determined using the 2^−ΔΔCt method. ΔΔCt was calculated using control group data.

Total RNA was isolated from AdMSCs using TRI reagent and RNA extraction kit (Zymo Research). First-strand cDNA was synthesized by using iScript RT-PCR mix (Bio-Rad) according to the user manual. SYBR green qPCR Master Mix (Bio-Rad), primers, and cDNA were mixed in a final reaction volume of 10 μL. Real-time PCRs were performed by using Applied Biosystems 7300 Fast Real-Time PCR System according to the manufacturer’s instructions. The oligonucleotide primers purchased from Integrated DNA Technology are listed in Supplemental Table 2.