Superscript vilotm

We extracted genomic DNA from paraformaldehyde-fixed paraffin-embedded (PFPE) odontoma sections using the WaxFree^TM Paraffin Sample DNA Extraction Kit (Trimgen, Sparks, MD, USA) according to the manufacturer’s instructions^{57 (link),58 (link)}. The mutational status of APC and CTNNB1 genes was investigated using PCR and direct sequencing as described previously^{59 (link)}. The PCR was performed for the mutation cluster region of the APC gene exon 15 (from codons 1274 to 1523) and the entire region of exon 3 of the CTNNB1 gene, respectively. The set of primers used for these genes was the same as that previously described^{59 (link)}.
Total RNA was extracted from PFPE sections using RNeasy FFPE Kit (Qiagen, Hilden, Germany)^{58 (link)}. RNA was successfully obtained in only 4 odontoma specimens, because of difficulty in purification from decalcified specimens. RNA was reverse-transcribed using SuperScript VILO^TM (Invitrogen) in order to prepare the first-strand cDNA. Each polymerase chain reaction (PCR) product (10 µl) was directly loaded onto 2% agarose gel, stained with ethidium bromide, and directly visualized under UV illumination. Primers for RT-PCR are listed in the Table S2.

RNA was extracted using an RNeasy mini kit (Qiagen). A SuperScript® VILOTM (Invitrogen/Life Technologies) cDNA synthesis kit was used to reverse transcribe cDNA from total RNA according to manufacturer's instructions. Quantitative PCR was performed using 7500 Fast Real-Time PCR detection system (Applied Biosystems). Reactions (25 μl each) were prepared in triplicate in a 96-well reaction plate. Each reaction contained 20 ng cDNA, 200 nM of each primer, 10 μl water and 12.5 μl Absolute Blue QPCR SYBR low ROX Mix (Thermo Scientific). DNA levels were normalized to the GAPDH calculated using a 2-ΔΔCt method. QPCR settings were as follows: Initialization at 95°C for 15 min, denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s and repeat for 40 cycles. Primers used for the qRT-PCR are listed below:
qRT-PCR F: 5′-AGT CGC ACA CTG CTA CAG GAC GA-3′
qRT-PCR R: 5′-GGC ACA AAG GCA TGT CGC ATG C-3′

Total RNA was isolated from transgenic and wild type flax plants using a total RNA isolation kit (Spectrum^TM, Sigma-Aldrich, St. Louis, MO, United States) and quantified. cDNA was synthesized from 2 μg total RNA according to manufacturer’s instructions (SuperScript^® VILO^TM, Invitrogen, Carlsbad, CA, United States) and used for cDNA preparation. To evaluate transcript accumulation, 1 μl of diluted cDNA mix was used as a template for the amplification of 67 bp nptII and 111 bp GUS fragments (Table 1). The PCR reaction mixture (25 μL) consisted of 2.5 μL 10× Taq buffer, 10 pM each of forward and reverse primer, 200 μM dNTPs, and 1 U of Taq DNA polymerase (Bangalore Genei, Bengaluru, India); 1 μL of cDNA was made up to a final volume of 25 μL with nuclease-free water. PCR amplification was carried out in a thermal cycler (Eppendorf, Hamburg, Germany) programmed with initial denaturation at 95°C for 4 min followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s (for both nptII and GUS gene RT-primers, Table 1) and extension at 72°C for 30 s. The final extension was carried out at 72°C for 7 min to amplify specific gene products. “Blank” consisted of nuclease-free water instead of cDNA, wild type contained 1 μL of cDNA of wild type, and positive control contained 25 ng of pCambia 2301 plasmid. The amplified gene products were analyzed on a 2.0% agarose gel.