Sybrgreen pcr mastermix

Total RNA from dental stem cells was isolated using GenElute Mammalian Total RNA Miniprep Kit (Sigma Aldrich, St. Louis, MO, USA). The RNA concentration was measured with a spectrophotometer (NanoQuant Infinite F200 PRO, TECAN). Total RNA (1 µg) was reverse-transcribed with MMLV RT kit (Evrogen, Moscow, Russia). Real-time PCR was performed with 50 ng cDNA and SYBRGreen PCR Mastermix (Evrogen, Moscow, Russia) using CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The thermocycling conditions were as follows: 95 °C for 5 min, followed by 45 cycles at 95 °C for 15 s, 60 °C for 30 s and 70 °C for 30 s (a 3-steps protocol is recommended by the PCR master-mix manufacturer). A final heating step of 65 °C to 95 °C was performed to obtain melting curves of the final PCR products. mRNA expression levels were calculated by the 2^−ΔΔCt method with the levels of gene transcription normalized to the housekeeping genes GAPDH encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ACTB encoding β-actin. Human blastocysts were used as a positive control to evaluate the quantity of OCT4 mRNA in dental cell cultures. The list of primers used for targeted genes amplification is shown in Table 1.

DNA extraction from leaves was performed using the cetyl trimethylammonium bromide (CTAB) protocol (Molecular Cloning, 3rd edition). Gene-specific primer pairs HOS1-1-target and HOS1-2-target (Supplementary Table S1) were used to amplify 381- and 372-bp long genomic regions containing the corresponding target sites, respectively. Obtained amplicons were ligated into a pJET (Thermo Fisher Scientific Inc., MA, USA) and sequenced using an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
To genotype mutations by a heteroduplex mobility assay (HMA), polyacrylamide gel electrophoresis (PAGE) analysis of amplicons subjected to denaturation/renaturation cycle was performed following the procedure of Reference [45 (link)]. To screen for mutations using high-resolution melting (HRM) analysis, we performed polymerase chain reaction (PCR)-HRM reactions with the same primer sets and 2.5 x SYBR green PCR master mix (Evrogen, Moscow, Russia) using CFX96 thermocycler (Bio-Rad Laboratories). Melting curves were determined by incubating the reaction mixes from 60 °C to 95 °C with an increment 0.2 °C for 10 s with a plate reading. Precision Melt Analysis software (Bio-Rad Laboratories) was utilized to discriminate between native and mutant HOS1 alleles.