Primerquest tool

Available full length chloroplast genomes of P. ginseng, P. quinquefolius and P. notoginseng were downloaded from Genbank. Chloroplast genomes were aligned using the Windows online version of MAFFT alignment program (https://bit.ly/3iqdjbm, 3 November 2021). Primer and probes were developed from diagnostic regions identified for each species (Tables S2 and S3). Primers and probes for both P. ginseng and P. quinquefolius were designed using the PrimerQuest^TM tool from Integrated DNA Technologies (IDT, Redwood City, CA, USA). Both assays were ordered from IDT (Redwood City, CA, USA) as PrimeTime™, qPCR probe assays, which contained primers and probe, pooled in a single vial. The primers and probe mix was suspended in 1 mL nuclease-free water to obtain a 10× concentration.

cDNA sequences were obtained from Genbank at the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov (accessed on 8 August 2019)). Primer sequences (Table S11 in Supplementary Material) were designed using the PrimerQuest^TM Tool from the Integrated DNA Technologies (IDT) website (https://www.idtdna.com/pages (accessed on 8 August 2019)). Sequence specificity was tested using the Basic Local Alignment Search Tool at NCBI [95 (link)]. Primers were obtained from IDT. Primer specificity was verified by melt curve analyses. All primers were designed to span exon/exon boundaries and thus exclude amplification of genomic DNA.

To further characterise the effects of TAK-242 in mitigating the
response of LPS, the expression of pro-inflammatory, matrix degradation and
ECM-related genes was examined at the end of the 24 h treatment period. Total
RNA was extracted from treated NP cells using the QIAGEN RNeasy kit following
manufacturer’s instructions. Concentration and purity were measured by
NanoDrop, with 260/280 ratios between 1.8 and 2.0 for all samples. 200 ng of
total RNA were converted to cDNA using the iScript cDNA Synthesis kit (BioRad).
Primers listed in Table 2 were designed
using the Integrated DNA Technologies PrimerQuest Tool (Integrated DNA
Technologies). Quantitative PCR was performed using QuantiStudio 6 Flex system
(Applied Biosystems, ThermoFisher Scientific) and iTaq Universal SYBR Green kit
(BioRad) with the following amplification protocol: 95 °C for 30 s
followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Analysis of gene expression was performed by QuantiStudio Real-Time PCR Software
v1.3 software (Applied Biosystems) using the 2^−
(ΔΔCt) method to calculate fold change relative to the
untreated group.

For gene expression analysis, the SVZ was excised and flash frozen. Total RNA was isolated from cell pellets using Trizol Reagent (Invitrogen) and the concentration was determined using both a BioPhotometer spectrophotometer (Eppendorf) and RNA 6000 kit (Agilent). cDNA was then generated from 1 μg of total RNA using Superscript II Reverse Transcriptase (Invitrogen). Quantitative real-time PCR reactions were run in 25-μL volumes on a CFX96 Fast Real-Time PCR Detection System (BIO-RAD) using iQ SYBR Green Supermix (BIO-RAD). Primers were designed using Primer-Blast (NCBI) and PrimerQuest Tool (Integrated DNA Technologies) and were validated for effective amplification without the interference of primer dimer up to a minimum 38 cycles. The following genes were evaluated: Nestin, Sox-2, Dcx, BDNF, Bmi-1, GAPDF (Supplemental methods). All qPCRs were performed in biological and technical triplicates. Data were analyzed using MATLAB and Microsoft Excel. GAPDH was used as an endogenous normalization control and the fold expression relative to GAPDH was determined by the ΔΔCt method. Relative gene expression between treated and untreated injured animals was evaluated using the method (Livak and Schmittgen 2001 (link)). All values were subtracted by 1 to show fold expression between the 2 cohorts from 0.

Total RNA was isolated from N. tabacum cells based on a modified protocol described by Toni et al. [34 (link)]. cDNA was synthesized from 100 ng total RNA using ReverTra Ace qPCR RT master mix (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. qPCR was carried out using SensiFast SYBR green master mix (Bioline, London, UK) and normalized to the endogenous EF1α and LF25 gene expression. The qPCR primer pairs were designed with PrimerQuest Tool (Integrated DNA Technologies, Coralville, IA, USA), targeting NtC4H (Accession No: MW260510), Nt4CL (Accession No: D43773), NtCHS (Accession No: KF927021), and NtCHI (Accession No: KJ730247) genes (Table 3). qPCR conditions were optimized for primer specificity, annealing temperature, and concentration. Each sample was tested in triplicate, and reaction mixtures were prepared according to the manufacturer’s instructions. qPCR assays were performed using the Applied Biosystems 7500 Real-Time PCR machine (ABI, Los Angeles, CA, USA) with the following cycling conditions: 50 °C for 2 min, 95 °C for 20 s, and run at 95 °C for 5 s, and 60 °C for 20 s for 40 cycles. The relative quantification of the gene expression level was calculated using ABI 7500 System Sequence Detection software v1.2 (ABI, Los Angeles, CA, USA).