Abi quantstudio 5 real time pcr system

Quantitative real-time PCR (RT-qPCR) was employed to validate the quality of RNA-seq results. Total RNAs from taproot samples at four stages including March, May, July, and November were extracted following the manufacturer’s instructions (Magen Biotech Co., Ltd., China) and reverse transcribed to cDNA following the manufacturer’s instructions (Vazyme Biotech Co.,Ltd., China). Each reaction was carried out using 10 μL 2× SYBR green reaction mix (Vazyme Biotech Co.,Ltd., China), 1.0 μL diluted cDNA, and 0.4 μL of each primer in a total volume of 20 μL system. RT-qPCR amplification reactions were performed in an ABI QuantStudio 5 Real-Time PCR System (Applied Biosystems). The specific primers used for RT-qPCR were designed with Primer3 web version 4.1.0 software, which are listed in Additional file 7: Table S3. Three technological replicates for each gene were performed and P.notoginsengYLS8 was used as an endogenous control. The relative gene expression value was calculated with the 2^−ΔΔCT method.

The RNA-seq results were then validated. Total RNA from P. notoginseng taproots from 1-, 2-, and 3-year-old plants was extracted using a kit (Magen Biotech Co., Ltd., China). A second kit (Tsingke Biotech Co., Ltd., China) was used to reverse transcribed the RNA to cDNA. An ABI QuantStudio 5 Real-Time PCR System (Applied Biosystems) was used to perform the RT-qPCR reactions. The primers used in the reactions were designed using Primer 3 web version 4.1.0, and are given in Table S1. The YLS8 gene was used as an endogenous control, and relative gene expression was calculated using the 2^−ΔΔCT method.

qRT-PCR was performed as described 48 (link), 49 (link), using ABI Power SYBR® Green master mix and detected by ABI QuantStudio 5 Real-Time PCR System (Applied Biosystems, Foster City, CA). The primer sequences are listed in supplementary Table S3.

5–10 oocytes collected were lysed in cell lysis buffer containing 0.2% Triton X-100 and RNase inhibitor after removal of zonal pellucida exposed to acidic M2 medium. The lysate was then reverse transcribed using SuperScript III First-Strand System (Invitrogen, 18080051) following manufacturers’ instructions. Quantitative PCR was conducted with One Step TB Green® PrimeScript^TM RT-PCR Kit (Takara, RR066A) on ABI QuantStudio5 Real-Time PCR system (Applied Biosystems). Murine Atcb was applied as an endogenous control since it remained stable during GV-MII transition detected by RNA-seq. Details of oligonucleotide primers used in this project were listed in Supplementary Data 7. Data were obtained from at least three replicated biological experiments per genotype with three technical repeats each time and were expressed as enrichment of 2^{-∆∆Ct49 (link)}.

Total RNA from human cells or tissues was extracted using Trizol reagent (Life Technologies) according to the instruction of the user manual. The concentration of RNA was determined by spectrophotometry at 260 nm using Nanodrop One (Thermo SCIENTIFIC). The purity of sample was determined by the 260/280 nm ratio. The detected ratio, ranging from 1.8 to 2.1, indicating that the extracted RNA was fine and suitable for subsequent experiments. cDNAs were generated from 1 μg RNA of each samples by using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). Real-time PCR (RT-PCR) analysis was performed using TB Green ™ Premix Ex Taq™ (Tli RNaseH Plus, Japan). A final volume of 20 μl mixture was amplified by RT-PCR using ABI QuantStudio-5 Real-Time PCR System (Applied Biosystems by Thermo Fisher Scientific). Reaction conditions were as follows: 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s. The relative mRNA expression was determined using the ΔΔCT method. The primer sequences used in real-time PCR are as follows: MCM2, forward 5’-CTACCAGCGTATCCGAATCCA-3’ and reverse 5’-CCTACAGCAACCTTGTTGTCCT-3’; NUP37, forward 5’-CTGCGTTTCGTGACCTTGTC-3’ and reverse 5’-TACACGTGCCAATGACCACA-3’; β-actin, forward 5’-CGACAGGATGCAGAACGAGA-3’ and reverse 5’-GACCCTGGATGTGACAGCTC-3’.