Goldenstar rt6 cdna synthesis mix

Total RNA was extracted from the samples using the Pure Plant Total RNA Extraction Kit (TSINGKE, Beijing) as described in the manufacturer’s instructions. The ratio of absorbancy at 260–280 nm was measured by Nanophotometer of Implen (Implen, Germany). RNA integrity was detected by agarose gel electrophoresis. All samples were stored at-80°C. All RNA samples served as templates for the cDNA synthesis by the Goldenstar RT6 cDNA Synthesis Mix(TSINGKE, Beijing). The reverse transcribed products was kept at −20°C.
Quantitative real-time PCR (qRT-PCR) was performed with Step One Plus real-time quantitative PCR instrument (Thermo Fisher, America) and using the SYBR Green I PCR master mix kit (TSINGKE, Beijing) according to the Cmanufacturer’s instructions. The relative amount of gene expression was calculated using the expression of actin as internal control gene. qRT-PCR primers were as following (Table 1). The relative quantity of gene expression was calculated using 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted from the stem samples at four stem swelling periods (stem diameters were 2 cm, 4 cm, 6 cm, and 8 cm) using the total RNA kit (Tiangen, Beijing, China). For cDNA synthesis, 1.0 µg RNA was reverse transcribed with oligo (dT) and random primers using the Goldenstar™ RT6 cDNA Synthesis Mix (TsingKe, Beijing, China). qRT-PCR was conducted with a Bio-Rad CFX96™ real-time PCR System (Bio-Rad, CA, USA) using 2 ×T5 Fast qPCR Mix (SYBRGreenI) (TsingKe, Beijing, China). All primers used are shown in Table S2. All experiments were performed with three independent RNA samples, and Actin2 (BjuB008540) was used as the internal control to normalize the gene expression. The relative mRNA expression was calculated using the 2^−ΔΔCT method (Pfaffl, 2001 (link)).

qRT-PCR utilized cDNA as a template. Amplification was performed with 2×TSINGKE Master qRT-PCR Mix (SYBR Green I) and GoldenStar™ RT6 cDNA Synthesis Mix (TSINGKE, China) on an Applied Biosystems ABI 7500 (Foster City, CA USA). Primers designed with Snap Gene (v4.3.6, Table S5) were used. Baseline and threshold cycles (Ct) were calculated by the system’s software. Data analysis used Actin expression for normalization.

The qRT-PCR was performed using cDNA templates. Each cDNA of the mRNA was amplified by qPCR using 2× TSINGKE Master qPCR Mix (SYBR Green I) and Goldenstar™ RT6 cDNA Synthesis Mix (TSINGKE, Beijing, China).
Applied Biosystems, Foster City, CA USA) using the SYBR-Green (Takara, Dalian, China) method. The primers were designed using SnapGene (version 3.5, Table S3). Each reaction was carried out with a volume of 20 μL, which contained 10 μL SYBR, 8.6 μL ddH₂O, 1 μL diluted template (1 μL of the generated first-strand cDNA diluted by 9 μL ddH₂O) and 0.2 μL of each of two gene specific primers. The following program was used conditions: 94 °C for 30 s (pre-denaturation) followed by 40 cycles at 94 °C for 30 s (denaturation), 60 °C for 20 s (primer annealing) and 72 °C for 43 s (extension and gathering the fluorescent signal). At the end, the melting curve analysis was executed for verifying the specificity of the primer with the following program: 95 °C/15 s, 60 °C/1 min, and 95 °C/15 s. The baseline and threshold cycles (Ct) were automatically determined by the own software of the system. Transcript levels were normalized against the average expression of the Actin gene.

Total RNA from endometrial tissues and ESCs was extracted using a Total RNA Extraction Kit (Solarbio, China). Goldenstar RT6 cDNA Synthesis Mix (Tsingke Biotechnology, China) and 2× T5 Super PCR Mix (Basic) (Tsingke Biotechnology, China) were used for reverse transcription and quantitative PCR, respectively. Relative FGFR2 expression was calculated by normalizing to β-actin using the 2^−ΔΔCq method (22). The primer sequences used were as follows: FGFR2 forward 5’-CCTGGATGTTGTGGAGCGAT-3’, reverse 5’-CTGTTACCTGTCTCCGCAGG-3’; β-actin forward 5’-GATTCCTATGTGGGCGACGA-3’, reverse 5’-CACAGGACTCC ATGCCCAG-3’.

Total RNA was extracted by an RNAprep FastPure kit (TSP413, Tsingke) according to the manufacturer’s instructions. Goldenstar RT6 cDNA Synthesis Mix (TSK134S, Tsingke) was used to synthesize cDNA from the RNA. The reactions were performed using 2 × T5 Fast qPCR Mix (SYBR Green I) (TSE202, Tsingke). The sequences of the primers are listed in Supplemental Table 1. The GAPDH gene was used as a reference gene. Changes in gene expression are shown as fold changes compared with the gene expression level in the controls.