Ssofast evagreen supermix kit

RNA was isolated from the dental pulp of the mandibular first molars from Ror2^fl/fl control mice and Osr2-Cre;Ror2^fl/fl mice at P3.5 using an RNeasy Micro kit (Qiagen). An iScript cDNA Synthesis kit (Bio-Rad) was used for cDNA synthesis. qPCR was then performed using an iCycler (Bio-Rad) with gene-specific primers, SYBR Green and an SsoFast EvaGreen Supermix kit (Bio-Rad). Values were normalized to Gapdh using the 2^−ΔΔCt method. The PCR primers are listed in Table S1.

Thirteen genes were selected for the validation of microarray results by RT-qPCR (Table 
1). The qPCR analysis was executed to conform to the qPCR MIQE guidelines
[42 (link)]. The validation experiments were performed using the same RNA samples that were used for the microarray study and other supplemental samples. Extracted RNA was converted into cDNA by reverse transcription of 500 ng total RNA using a Quantitect cDNA synthesis kit (Qiagen). Then, qPCR assays were carried out using SsoFast Evagreen Supermix kit (Bio-Rad, Hercules, CA) and gene-specific primers (250 nM) on a CFX96 rapid thermal cycler system (Bio-Rad). The cycling conditions were: 3 min of polymerase activation at 98°C followed by 40 cycles at 98°C for 2 s and 57°C for 5 s. Melting curves were generated after each run to confirm a single PCR product.
Primers (Integrated DNA technologies, Coralville, IA) that were used for detection of genes were all verified to have reaction efficiency between 90-110% (Table 
1). The GeNorm applet v.3.5 (http://medgen.ugent.be/~jvdesomp/genorm/) was used to initially determine the two most stable reference genes from a set of six reference genes using random samples from the cDNA panel generated for the qPCR validation of the microarray. Therefore, normalization of the data was done using the reference genes Hypoxanthine (Hipox) and Peptidylprolyl Isomerase A (Ppia).

Total RNA was extracted from MSCs using Trizol reagents (Invitrogen Life Technologies) according to manufacturer’s instructions. 1 μg of total RNA from each sample was reverse-transcribed into cDNA using a reverse transcription kit (Thermo Scientific). Real-time RT-PCR was performed using SsoFast EvaGreen Supermix kit (Bio-rad) with GAPDH as a reference control. Reactions were carried out at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Primers used for real-time RT-PCR were as follows: CXCR4-F, 5′-GAAGTGGGGTCTGGAGACTAT; CXCR-R, 5′-TTGCCGACTATGCCAGTCAAG; GAPDH-F, 5′-TGACAACTTTGGCATCGTGG; GAPDH-R, 5′-TACTTGGCAGGTTTCTCCAGG.

Total RNA was isolated from peach tissue samples and Arabidopsis by using a modified cetyltrimethylammonium bromide (CTAB) method (Liu et al., 2015 (link)) and the TRIzol Reagent Kit (Ambion, Hopkinton, MA, United States), respectively. First-strand cDNA was synthesized with HiScript^® II Q Select RT SuperMix (Vazyme) after removal of genomic DNA contamination by TURBO DNase (Ambion) following the manufacturer’s protocol. Reverse transcription quantitative PCR (RT-qPCR) was performed using the SsoFast EvaGreen Supermix kit (Bio-Rad, CA, United States) with the CFX96 instrument (Bio-Rad, CA, United States) according to the following program: 95°C for 3 min, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s and then 95°C for 10 s followed by a continuous increase from 65°C to 95°C at the heating rate of 0.5°C/s for dissociation curve analysis (Zhao et al., 2017 (link)). The PpTEF2 (GenBank accession: No. JQ732180) and ATACT2 (GenBank accession: No. AT3G18780) genes were used as the internal reference genes to normalize expression values for peach and Arabidopsis, respectively (Tong et al., 2009 (link); Zhou et al., 2013 (link)). The primer sequences for RT-qPCR were designed with Primer Premier 5 and listed in Supplementary Table 1.

Total RNA was isolated from cultured cells using the RNeasy Mini Kit (74106, Qiagen, Hilden, Germany). Total RNA (2 μg) was reverse transcribed by the iScript Select cDNA Synthesis Kit (170–8896, Bio-Rad, Hercules, CA, USA) in accordance with the manufacturer’s instructions. Using 1/10 of the cDNA sample and independent qPCR primers targeting transcript of the indicated genes, qPCR was performed by the SsoFast EvaGreen Supermix Kit (172–5200, Bio-Rad) in accordance with the manufacturer’s instructions. Primer sequences used for RT-qPCR are described in Supplementary Table 1. Expression of the respective target transcript was quantified using qRT-PCR and normalized to β-actin and HPRT. Data are presented as fold-change relative to vehicle control. Data are mean±s.d. of experiments in triplicate and representative of three independent experiments.

To study mRNA expression of different genes in hd2 mutants and HD2 overexpression lines, quantitative real time PCR (RT-qPCR) was performed. RNA was isolated from leaves or seedlings using the Plant/Fungi Total RNA Purification Kit (Norgen) and cDNA was synthesized using iScript Reverse Transcription Supermix (Bio-Rad). The RT-qPCR was performed using a SsoFast EvaGreen Supermix kit (Bio-Rad) on CFX96 Real-time PCR detection system (Bio-Rad) following the manufacturer’s instructions. Data were analyzed using Bio-Rad CFX Manager 3.1 software. The expression levels were normalized to the ACTIN2 gene. The ΔΔCT method was applied to calculate fold change in the expression level (Livak and Schmittgen, 2001 (link)). Three independent RT-qPCR analysis were performed with three technical replicates for each biological replicate. All primers used for RT-qPCR analysis are listed in Supplementary Table 3.