Abi quantstudio 6 flex system

Total RNA was isolated from GC cell lines or GC tissues with RNAiso Plus reagent (TaKaRa, Japan) based on the manufacturer's protocol. The PrimeScriptTM RT Reagent Kit with gDNA Eraser, obtained from TaKaRa, Japan, was used to synthesize the first strand cDNA. Quantitative real‐time PCR (qRT‐PCR) was performed with the SYBR green reagent (TaKaRa, Japan) on ABI QuantStudioTM 6 Flex system (Thermo Fisher Scientific). Relative quantification of PCR products was performed with the comparative Ct method (2^−ΔΔCt), and β‐actin was used an endogenous control. The results were all repeated three times. The following primer pairs were used to amplify and measure the amount of PIK3CD, 5′‐CATATGTGCTGGGCATTGGC‐3′ and 5′‐TTTCACAGTAGCCCCGGAAC‐3′). The primers for β‐catenin: 5′‐ CCTGGCACCCAGCACAATG‐3′ and 5′‐ GGGCCGGACTCGTCATACT‐3′).

The total RNAs were extracted from cultured GC cells or GC tissue specimens with Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions, and then the concentration of RNA was quantified by a Fisher Scientific NanoDrop™ 2000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, USA). RNA was reverse-transcribed into cDNA using the PrimeScript™ RT reagent kit (Takara Japan). cDNA was then used to perform Standard qRT-PCR analysis by ABI QuantStudioTM 6 Flex system (Thermo Fisher Scientific) using SYBR green reagent (TaKaRa, Japan) in a final volume of 10 μL. Relative mRNA expression of SP1 and PIK3CB were determined by 2^-ΔΔCt method using their threshold cycle (CT) values and β-actin was considered as an internal control. The following primers for gene amplification were used:
SP1-Forward: 5′-TGGCAGCAGTACCAATGGC-3′; SP1-Reverse: 5′-CCAGGTAGTCCTGTCAGAACTT-3′; PIK3CB-Forward: 5′-CCTCTCAGAACTCTATGTTGAA-3′; PIK3CB-Reverse: 5′-ATCCTGTCGTAAATCATCAC-3′; β-actin-Forward: 5'-CCTGGCACCCAGCACAATG-3'; β-actin-Reverse: 5'-GGGCCGGACTCGTCATACT-3'.

Total RNA from the HCC cell lines was isolated with Trizol reagent (Sigma Aldrich, MO, USA) according to the manufacturer’s protocol. A Fisher Scientific NanoDrop™ 2000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA) was used to investigate the quality and concentration of RNA, and 1 μg total RNA was used to synthesize cDNA using the PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan) prior to qPCR analysis. SYBR green reagent (TaKaRa, Japan) was used to perform qPCR analysis on an ABI QuantStudio^TM 6 Flex system (Thermo Fisher Scientific, Inc.). β-actin was used as an internal control, and relative mRNA expression level was quantified using the 2^−ΔΔCt method. The qPCR primers used in this study were included in Supplementary Table 2.

Total RNA from hGMSCs, which were treated with uninduced, osteogenic induced, or osteogenic induced with SB431542 medium, was extracted with the trizol reagent kit (Invitrogen, Carlsbad, CA, USA). And cDNA was synthesis with Hiscript RT supermix kit (Vazyme; Nanjing, China) according to the manufacturer’s instructions. cDNA amplification was used by SYBR qPCR master mix (Vazyme), and qRT-PCR was performed by ABI QuantStudio™ 6 Flex system (Thermo Fisher Scientific, MASS, USA). All qRT-PCR reactions were performed in triplicate in each experiment. The BMPs genes (BMP2 and BMP4) forward and reverse primers are listed in Additional file 1: Table S1. The results were analyzed using the 2^−ΔΔct method and normalized by β-ACTIN.

Total RNAs were extracted from adipose tissues using Trizol reagent (Invitrogen, USA) and reverse-transcribed into cDNA using reverse transcription Kit (R323-01, vazyme, China) according to the manufacturer's instructions. RT-PCR was performed with a ChamQ Universal SYBR PCR Master Mix (Q711-02, vazyme, China) by ABI QuantStudio™ 6 Flex system (Thermo Fisher Scientific, MASS, USA). The specific primers are listed in Table 1. The mRNA expression was relatively quantified according to CT value and calculated by 2^−∆∆Ct method.Table 1

Primers used for quantitative real-time PCR analysis

Primer name	Species	Sequence forward (5'to3')	Sequence reverse (5'to3')
IL10	Mouse	GCTGAGGCGCTGTCATCGATTT	GGCCCTGCAGCTCTCAAGTGT
IL6	Mouse	TCAATTCCAGAAACCGCTATGA	CACCAGCATCAGTCCCAAGA
IL1β	Mouse	CGTGCTGTCGGACCCATATGAG	GCCCAAGGCCACAGGTATTT
IL4	Mouse	TCACTGACGGCACAGAGCTA	CTGTGGTGTTCTTCGTTGCTG
TNFα	Mouse	ACGTCGTAGCAAACCACCAA	ACCCTGAGCCATAATCCCCT
PPARγ	Mouse	TCGCTGATGCACTGCCTATG	GAGAGGTCCACAGAGCTGATT
C/EBPα	Mouse	CAAGAACAGCAACGAGTACCG	GTCACTGGTCAACTCCAGCAC
SREBP-1c	Mouse	GAGCGAGCGTTGAACTGTAT	ATGCTGGAGCTGACAGAGAA
Adiponectin	Mouse	TGTTCCTCTTAATCCTGCCCA	CCAACCTGCACAAGTTCCCTT
GAPDH	Mouse	ACAACTTTGGCATTGTGGAAG	GTTGAAGTCGCAGGAGACAAC

Total RNAs were extracted from control and SPc nanoparticles exposed third instar larvae using TRIeasy (Yeasen Biotech, Shanghai, China). The cDNAs were synthesized by the Hifair First Strand cDNA Synthesis Kit (Yeasen Biotech) and used as templates for PCR experiments using the Perfect Start Green qPCR Super Mix (TransGen Biotech, Beijing, China). qRT-PCR were conducted on an ABI QuantStudio 6 Flex System (Thermo Fisher, USA). The Rpl32 gene was used as internal control for qRT-PCR, and the gene expression level was examined by the ΔΔCt method [88 (link), 92 (link)]. The primers used for qRT-PCR in this study are listed in Additional file 6: Table S2.