Goldenstar rt6 cdna synthesis kit ver 2

Based on the results of SQANTI analysis, Astalavista software was used to ascertain alternative splicing events. The main types of alternative splicing include intron retention (IR), exon skipping (ES), alternative 5′ splice sites (A5), alternative 3′ splice sites (A3), and mutually exclusive exons (MXE). To validate the detected AS events, two unigenes were randomly selected for validation using male-induced cannabis flowers (IMF). Primers were designed for selected unigenes using Primer 5.0 software (Table S1). Total RNA from the male-induced flowers was extracted as described above. The Goldenstar^TM RT6 cDNA Synthesis Kit Ver.2 (TsingKe, China) and 2×Es Taq MasterMix (Dye) (Cowin, China) were used for reverse transcription and PCR assays, respectively. The PCR amplification conditions were as follows: 94 °C for 1 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min (30 cycles), and 72 °C for 3 min. PCR products were detected using 1% agarose gel electrophoresis. The FastPure^® Gel DNA Extraction Mini Kit (Vazyme, China) was used for gel recovery, and the pEASY^®-Blunt Cloning Kit was used to ligate the recovered product to the B vector. This was sequenced to confirm whether the amplified sequences were consistent.

A total of 35 breast cancer tissues and 35 adjacent normal tissues were collected. This study was approved by the Ethical Committee of Hospital and was conducted in accordance with the Declaration of Helsinki. In addition, each patient offered written informed consent. All tissues were immediately stored at −80°C.
Total RNA of tissue was extracted using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. Then, the extracted RNA was qualitatively controlled and quantified by Nanodrop. The equivalent amount of RNA was reverse transcribed into cDNA with Golden star TM RT6 cDNA synthesis KIT ver.2 (TsingKe, Beijing, China). LncRNA and mRNA expression was then examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) with Master qPCR Mix (SYBR GREEN 1) (TsingKe, Beijing, China) and Biosystems 7,500 Fast Real-time PCR System. GAPDH expression was used as endogenous control. The primers were designed using Primer 5.0 software (Table 2). The 2^–ΔΔCt method was used to calculate the levels of lncRNA expression and mRNA expression. Excel was used to analyze the qRT-PCR data, and each reaction was performed in triplicate. Two groups were performed using t-test (p < 0.05).

Total RNA was extracted using Polysaccharide polyphenol Plant total RNA Extraction Kit (PD Biotech, Shanghai, China), and cDNAs were synthesized using Goldenstar^® RT6 cDNA Synthesis Kit Ver.2 (TSINGKE, Beijing, China). Real-time PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on an ABI prism 7500 real-time PCR System. Mungbean VrACTIN3 (Vradi03g00210) and Arabidopsis ACTIN2 (AT3G18780) were selected as reference genes to normalize the expression data. The 2^−ΔCT or 2^−ΔΔCT methods were used to analyze relative expression levels. The results were calculated from three biological replicates and three technical replicates. Statistical analysis and plot drawing were conducted using Excel and GraphPad Prism 5 software. Heat maps were drawing by TBtools after the FPKM value taken logarithm (LOG 2). The primers used in this study are listed in Table S2.

By the manufacturer's instructions, the total RNA was extracted from culture cells or tissues using the Trizol Reagent (Ambion, USA). The first-strand cDNA was synthesized by the Goldenstar™ RT6 cDNA Synthesis Kit Ver 2 (Beijing TsingKe Biotech Co., Ltd., China). Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was performed using the SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd., China) on a ViiA7 real-time PCR (Applied Biosystems, Foster City, CA, USA). The reaction conditions are as follows: predenaturation 95°C to 15 s; 95°C to 15 s, 55°C 30 s, 72°C 45 s; melting curve. The whole qPCR reaction lasted 40 cycles. All RT-qPCR primer sequences were designed and synthesized by Beijing TsingKe Biotech Co., Ltd. (Beijing, China). The primers used in this study were the following: BTK, forward 5′-GTCAGAGACTCCAGCAAAGCTG-3′ and reverse 5′-TACTGGCTCTGAGGTGTGGAAC-3′, GAPDH, forward 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

To verify the accuracy of the RNA-Seq data and assay the expression levels of the SMV-CP gene, qRT-PCR assays were conducted using gene-specific primers. Semi-quantitative RT-PCR was also used to detect the expression of viral coat protein genes according to Zhang et al., 2019 [40 (link)]. Total RNA from the same treated samples was extracted from V1 leaves in soybeans. Reverse transcription was performed using a reverse transcription kit from Beijing Tsingke Biotech Co., Ltd. (Goldenstar™ RT6 cDNA Synthesis Kit Ver.2). In addition, 2×RealStar Fast SYBR qPCR Mix (Tsingke Biotech Co., Ltd., Beijing, China) was used, and an Eppendorf Mastercycler ep realplex (Eppendorf, Hamburg, Germany) instrument was used for the qRT-PCR experiment. Each treatment contained three independent biological replicates and three technical replicates. The expression level of the soybean β-actin gene was used as an internal reference. The fold change value of gene expression was calculated using the 2^−∆∆Ct method. The sequences of specific primers are listed in Tables S2–S4.

Ten-day-old seedlings from Col-0 and two lon1 mutant lines, grown under long-day conditions, were collected for RNA extraction. The collected seedlings, approximately 0.1 g in weight, were rapidly frozen in liquid nitrogen and then ground into a fine powder using 2 mm beads and a homogenizer. RNA was extracted using the TaKaRa (9769) MiniBEST Plant RNA Extraction Kit (Shiga, Japan), following the manufacturer’s instructions. For cDNA synthesis, 500 ng of RNA was utilized, employing the TSINGKE (TSK302M) Goldenstar™ RT6 cDNA Synthesis Kit Ver.2 (Beijing, China). Transcripts of selected genes were quantified using TaKaRa (RR420A) TB Green^® Premix Ex Taq™ (Tli RNaseH Plus) (Shiga, Japan) and the Realplex2 system from Eppendorf (Hamburg, Germany). The Q-PCR data were normalized to housekeeping genes, specifically UBQ10, before making comparisons across different ecotypes. All primers used for Q-PCR analyses are listed in Supplementary Data S8.