Quantstudio design and analysis software

Once miRNAs had been extracted from all serum samples, the four selected miRNAs (Hsa-miR-497-5p, Hsa-miR-155-5p, Hsa-miR-423-5p and Hsa-miR-365-3p), as well as the housekeeping (Hsa-miR-93-5p) and Cel-miR-39, were determined using RT-qPCR. Hsa-miR-93-5p was chosen as housekeeping for its widely regarded potential as a reference gene^{21 (link),22 (link)} and because in our previous study it was revealed to be a reference miRNA candidate by the GeNorm algorithm in the profiling stage for our sample pool^{13 (link)}. We used 2 μl of each miRNA sample for retro-transcription (RT) in 6 μl of total volume reaction using the TaqMan Micro RNA Reverse Transcription kit (ThermoFisher) and specific primers for each miRNA contained in the TaqMan miRNA Assay kit (ThermoFisher). Amplification of the RT product was performed using TaqMan Universal PCR Master Mix II (ThermoFisher) and the specific primers and probe for each miRNA present in the TaqMan miRNA Assay kit (ThermoFisher). The reaction was carried out using QuantStudio 5 Real-Time PCR System (ThermoFisher) in 384-well plates, and amplification curves were analyzed with QuantStudio Design and Analysis Software (v1.5.1; ThermoFisher).

The data analysis was performed using the analysis module of QuantStudio™ Design and Analysis software, v1.5.1 (Thermo Fisher Scientific) using the standard curve generated as described above. Alternatively, the analysis can be performed using the formula below, where Cq is the quantitation cycle and Yintercept as well as the slope are derived from the calibration curve (Fig. 1D). An example of such a calculation is provided in the Supplementary Information. $Quantityperwell=\frac{Cq-Yintercept}{slope}$

Total RNA was isolated from either HeLa or HEK293T cells by using TriZol-reagent and the manufacturer protocol (Invitrogen). One microgram of total RNA was converted to cDNA by using reverse transcriptase (RT)-PCR according to the manufacturer’s protocol (qPCRBIO cDNA Synthesis Kit; qPCR Biosystems). Real-time RT-PCR was performed using qPCRBIO SyGreen Blue Mix (qPCR FBiosystems) to detect mRNA expression of ubiquitin genes. The list of primers is provided in Supplementary Table 2. The real-time PCR data was analyzed using QuantStudio™ Design and Analysis Software (Thermo Fisher).

Real-time reverse transcription followed by quantitative PCR (RT–qPCR) was done essentially as described by López-Dolz et al. (2020) (link) in a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Primers used for RT–qPCR are listed in Supplementary Table S1. Target RNA expression levels were calculated relative to two S. lycopersicum reference genes (SlACT and SlEXP) using the delta delta cycle threshold comparative method of the QuantStudio Design and Analysis Software (version 1.5.1.; Thermo Fisher Scientific).

The kits: FastQuant RT Kit with gDNase (TIANGEN Biotech Co., Ltd., Beijing, China); SuperReal PreMix Plus (SYBR Green) (TIANGEN Biotech Co., Ltd., Beijing, China) were used for the analysis of samples.
RNA Samples subjected to RNA-seq analysis used the FastQuant RT Kit with gDNase (TIANGEN) for first-strand cDNA synthesis. Five sequences for the RNA-seq consistency test were randomly selected from the differentially expressed gene data obtained. In this case, 12 unique up-expression consistency test sequences were from the gene data obtained by RNA sequencing of Wuzhishan strains. The NCBI primer-blast program was used to design all of the quantitative PCR primers. Table 4 provides some background information. Beijing Qingke Biotechnology Co., Ltd. (Beijing, China) performed the primer synthesis. The Actin gene, which was stably expressed in all RNA-seq samples and was highly expressed, was selected as the reference gene [34 ]. The real-time PCR test uses SuperReal PreMix Plus (SYBR Green). The reaction system consisted of 20 μL, including 1 μL of cDNA template (100 ng/L), and 0.75 μL for front and back primers. Data analysis was performed using QuantStudio ™ Design and Analysis Software (version 1.5.1, Thermo Fisher Scientific). For qRT-PCR data, relative expression log₂FC was calculated using the Ct method and compared with RNA sequencing data. Each sample had three replicates.

There is no reliable method to quantify RNA molecules in bodily fluids. In this study, we established an absolute quantitative method to quantify plasma tRF-27 levels. The stem-loop-structure containing the tRF-27 sequence was cloned into the pUC57 plasmid (General Biology, Hefei, China) to prepare the recombinant plasmid pUC57-tRF-27. To prepare high-concentration standards, the lyophilized plasmid was dissolved in 50 μL of DNase-free water, and eight dilution gradients were prepared for qRT-PCR. The Applied Biosystems™ QuantStudio™ 3 Quantitative Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) coupled to QuantStudio™ Design and Analysis Software was used to detect the quantification cycle (C_q) value. Based on the copy number of the template and the C_q value of qRT-PCR, a standard curve of the tRF-27 copy number was generated. According to the C_q value and the standard curve formula, the absolute amount of tRF-27 in plasma was calculated.