Taq pro universal sybr qpcr master mix kit

Total RNA from A549 cells, A549/DDP cells, A549-exo, and A549/DDP-exo was extracted using Trizol™ reagent (15596026; Invitrogen) and Total exosomal RNA & Protein isolation kit, and the concentration of total RNA in each sample was determined using a NanoDrop-1000 ultra-micro spectrophotometer (Thermo Fisher). Reverse transcription and amplification of total RNA were performed to detect miR-424-5p expression, using Bulge-Loop miRNA qRT-PCR Starter Kit (C10211-1; Ribobio, China). TP53, SOCS5, and SOCS6 were detected using FastKing RT Kit (KR116; Tiangen, Germany) to perform reverse transcription, and Taq Pro Universal SYBR qPCR Master Mix Kit (Q712-03; Vazyme, China) to perform amplification. Primer sequences for U6, GAPDH, miR-424-5p, SOCS5, SOCS6, and TP53 are shown in Supplemental Table 2.

RNA isolation was performed using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (RC112-01, Vazyme, China) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from the isolated RNA using the HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (R333-01, Vazyme, China). Real-time PCR was carried out on the synthesized cDNA using the Taq Pro Universal SYBR qPCR Master Mix Kit (Q712-02, Vazyme, China) in real-time PCR system (ABI7500, Applied Biosystems, USA). The mRNA levels of target genes were quantified using the 2^−ΔΔCT method. Please refer to Additional file 1: Table S1 for the specific primers utilized in the analysis.

RNA isolation was performed using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from the isolated RNA using the HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (Vazyme, Nanjing, China). Real-time PCR was carried out on the synthesized cDNA using the Taq Pro Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China) in an ABI7500 real-time PCR system (Applied Biosystems). The primer sequence was from the previous references (15 (link)).

Based on the LD, all genes within the 40 kb range of significant sites were identified using the reference genome of Zhongmu-4. The orthologues were identified by comparing them with the A. thaliana genome. The candidate genes were identified by combining the GWAS and transcriptomic results. The root tips of the plants were collected and the RNA was extracted using the MiniBEST RNA kit (TaKaRa, Beijing, China), according to the manufacturer’s instructions, and washed with the DNA incubation solution provided with the kit to remove the residual DNA in the genome. In total, 60μL RNA was extracted from each sample and used to construct the cDNA library. A quantitative real-time PCR (qRT-PCR) was constructed using the Taq Pro Universal SYBR qPCR Master Mix kit (vazyme, Nanjing, China) on the CFX96 Touch™ RT-PCR system (BioRad, Los Angeles, CA, USA). Three technical replicates were set for each sample. All primers used in this study are listed in Table S10, and the alfalfa actin gene was used for an internal control. The data were quantified with the 2^−(ΔΔCT) method [54 (link)]. The SPSS 26 version software was used for the analysis of variance (ANOVA). Data visualization was presented using the Origin 2019b version software.

The expression patterns of YABBY genes were analyzed by using RNA-seq data from different tissues of M. dodecandrum. After standardizing the data, TBtools was used to draw the heatmap of MdYABBYs [31 (link)]. To further analyze the expression patterns of the YABBY gene family in M. dodecandrum, RT-qPCR was performed. The RT-qPCR primers were designed by Primer3Plus (https://www.primer3plus.com/, accessed on 17 November 2022); Supplementary Table S1). A Taq Pro Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China) was used for the RT-qPCR analysis. The MdActin gene was used as an internal reference [32 (link)], and the biological repetition and technical repetition were performed three times. The expression level was calculated by the 2^−∆Ct method [1 (link),32 (link)].

The total RNA of C. elegans samples was isolated using the RaPure Total RNA Kit (Magen, Shanghai, China) following the manufacturer’s protocol. In brief, the RLT lysis buffer was used to lyse the worms, and then total RNA was collected and purified using HiPure RNA Mini Columns. Finally, the RNA was dissolved with RNase free water. The purity and concentration of RNA were determined using a NanoDrop spectrophotometer (Thermo Fisher, Waltham, MA, USA). cDNA was synthesized using the HiScript III. All-in-one RT SuperMix Perfect for qPCR Kit (Vazyme, Nanjing, China). Relative quantitative RT-PCR was performed using Rotor-Gene Q (Qiagen, Beijing, China) with a Taq Pro Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China). RT-PCR amplification conditions included denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s, and extension for 32 s at 72 °C. Act-1 was used for normalization. All primer pairs were synthesized by Synbio Technologies (Jiangsu, China), and the sizes of PCR products were between 100 to 300 bp. The primer sequences used are listed in Table 6. Results were analyzed using the comparative threshold cycle (Ct) method and expressed as the fold change in gene expression (2^−ΔΔCt).

The HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) kit was used for reverse transcription. Furthermore, Taq Pro Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China) was used for RT-qPCR in the Bio-Rad iCycler iQ system (Bio-Rad, USA). RT-qPCR was carried out under the following conditions: 95 °C for 30 s, then 40 cycles (95 °C for 10 s and 60 °C for 30 s). The RT-qPCR primer sequences used for β-actin, Prkcg, Pik3r1, Akt1, Stat3, Rela, Il4, Il12a, Cd68, and Ptprc (B220) are shown in Table 2. The relative level (fold change (2^−ΔΔCT) or log2 fold change) for each transcript was calculated. The experiment was performed in triplicate and repeated three times.