Hieff qpcr sybr green master mix high rox plus

The RNA-seq data was confirmed by qRT-PCR analysis of fifteen DEGs selected randomly. cDNA was synthesized from 1 μg of total RNA by Evo M-MLV RT Mix Kit with gDNA Clean for qRT-PCR [AG11728, Accurate Biotechnology (Hunan) Co., Ltd], then analyzed by qRT-PCR using Hieff^® qPCR SYBR^® Green Master Mix (High Rox Plus) (Cat No. 11203ES08; Yeasen, Shanghai, China) on StepOnePlus system (Applied Biosystems, United States). BrActin (Bra028615) was used as a quantitative reference (Dheda et al., 2004 (link)). Primer 6.0 software (Premier, Ottawa, Canada) was used to design the corresponding primers, which are listed in Supplementary Table 3. Each 20 μL PCR reaction comprised 10 μL of 2× SYBR Green Master Mix, 0.4 μL of 10 μM primers, 2 μL of cDNA template, and 7.2 μL of ddH₂O. qRT-PCR was as follows: 95°C 5 min, 95°C 10 s, 60°C 30 s, 40 cycles. Each sample had three biological and three technical duplicates. The 2^–ΔΔCt approach was applied to quantify relative gene expression levels (Schmittgen and Livak, 2008 (link)).

Total RNA of the cells was extracted using MolPure® Cell/Tissue Total RNA Kit (YEASEN, China). cDNA was synthesized using Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (YEASEN, China). qPCR was performed using Hieff® qPCR SYBR Green Master Mix (High Rox Plus) (YEASEN, China) on ABI 7500. Primers sequences for the detection of IL4I1 were 5’-CGCCCGAAGACATCTACCAG-3’ (forward) and 5’-GATATTCCAAGAGCGTGTG CC-3’ (reverse). The primer of Gapdh was 5’-GCAGGGGGGAGCCAAAAGGG-3’ (forward) and 5’- TGCCAGCCCCAGCGTCAAAG-3’ (reverse).

Total RNA was extracted from cells via RNA Express Total RNA Kit (M050, NCM Biotech, China), and the reverse transcription of RNA was conducted by the Revert Aid First Strand cDNA Synthesis Kit (K1622, Thermo Scientific, United States). Following, the RT-qPCR was executed by Hieff qPCR SYBR Green Master Mix (High Rox Plus) (11203ES, YEASEN Biotech Co., Ltd, China). The 2^-ΔΔCT method was selected to evaluate the expressions of the included genes. The primer sequences are presented in Table S2.

Following the manufacturer’s guide, total RNA was extracted from GC cells using an RNA-Quick Purification Kit (RN001, Esunbio, Shanghai, China). The extracted RNA was used to reverse transcribed to the corresponding cDNA using Hifair^® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen, Shanghai, China), and qRT-PCR analysis was performed using the Hieff^® qPCR SYBR Green Master Mix (High Rox Plus)(Yeasen, Shanghai, China). Thermal cycling conditions were as follows: denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 sec, and extension at 60°C for 30 sec. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control and the results were normalized to its expression. Fold changes in mRNA expression were calculated using the comparative Ct method (ΔΔCt). Primer pairs for target genes used in the qRT-PCR assay are listed in Table 1.

Total cellular RNA was drawn from cell lines utilizing RNA Express Total RNA Kit (M050, NCM Biotech, China). Next, total RNAs were used for cDNA synthesis with Revert Aid First Strand cDNA Synthesis Kit (K1622, Thermo Scientific, United States). Subsequently, the expression level of each gene was quantified by Hieff qPCR SYBR Green Master Mix (High Rox Plus) (11203ES, YEASEN Biotech Co., Ltd, China) and calculated with the 2-^ΔΔCT method. GAPDH was used as the internal reference for normalization. The primer sequences are listed in Table S3.

All the procedures, like RNA extraction, reverse transcription, and RT-qPCR, were conducted as per the instructions of the kit. Firstly, the total RNA of cells was extracted using SteadyPure Quick RNA Extraction Kit (Accurate Biotechnology (Hunan) Co.,Ltd). Then, the RNA reverse transcription was performed utilizing Hifair III 1st Strand cDNA Synthesis SuperMix for RT-qPCR (Yeasen Biotechnology (Shanghai) Co.,Ltd). Finally, the Hieff qPCR SYBR Green Master Mix (High Rox Plus) (11203ES, YEASEN Biotech Co., Ltd, China) was used for RT-qPCR and the 2^{^-ΔΔCT} method was applied to calculate the relative gene expression level. Supplementary Table 1 displays the sequences of primer used in the present study.

Total RNA was extracted from the placenta and HTR-8/Svneo cells using TRIzol (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The quantity of RNA was examined using a NanoDrop2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). For mRNA detection, total RNA (2 μg) was used for cDNA synthesis using a Hieff^® UNICON qPCR SYBG Green Master Mix (High Rox) (Yeasen Biotechnology, Shanghai, China). Gene expression was assessed by qPCR with 2 μL of the synthetized cDNA using a Hieff^® qPCR SYBR Green Master Mix (High Rox Plus) (Yeasen Biotechnology). The qPCR reactions were performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). GAPDH was set as the normalizing control. Relative quantities were calculated using the 2−△△CT method. The sequences of all primers used are listed in Table 1.