Superscript 3 first strand synthesis system for rt qpcr

Total RNA, collected from 800 antennae, 80 de-antennaed heads, 500 legs, 500 cornicles-cauda and 40 remaining body parts of wingless adult aphids and from 30 aphids of each different nymphal instar (I, II, III, IV) and each adult morph, was extracted using TRI Reagent (Sigma, St. Louis, MO, United States), following the manufacturer’s instructions. The concentration of total RNA was measured spectrophotometrically at 260 nm, using a NanoDrop ND-1000 instrument (Nanodrop Technologies, Wilmington, DE, United States). The purity of RNA was estimated at absorbance ratios OD260/280 and OD260/230, and the integrity was verified on 0.8% agarose gel electrophoresis. In order to efficiently remove genomic DNA contamination, the samples were treated with 1U of DNase I (Deoxyribonuclease I, Amplification Grade, Invitrogen-Life Technologies, Carlsbad, CA, United States) per microgram of RNA for 15 min at room temperature, following the manufacturer’s guidelines. cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-qPCR (Invitrogen-Life Technologies), according to the manufacturer’s protocol, using 5 μg of total RNA per sample. The cDNA synthesis reaction was diluted with nuclease-free water to a final volume of 100 μl and immediately used for RT-qPCR studies or stored at -20°C.

MC3T3-E1 cells were seeded at a density of 2 × 10⁵ cells/100 mm dish and cultured in α-MEM-containing 10% FBS. After incubation for 2 d, the growth medium was changed to 2% FBS, 100 μg/mL LF-II, 50 μg/mL ascorbic acid, and 10 mM β-GP. Cells were cultured for 1 week before 0.2 mM HA was added for 2 or 8 h. Next, total RNA was extracted using the SuperScript III first-strand synthesis system for RT-qPCR (Invitrogen, CA, USA). Osteocalcin, runx2, and osterix mRNA expression were assessed using a LightCycler 96 system (Roche Diagnostics K.K., Tokyo, Japan). Their relative expression was normalized to β-actin as the housekeeping gene. The oligonucleotide primers for the amplification of related genes were designed and checked using Primer-BLAST (NCBI, NIH, MD, USA), as listed in Table 2.

Total RNA was extracted from leaves of teak and tobacco transgenic lines using the TRIzol reagent (Thermo Fisher, Waltham, MA, USA) to examine the TgNAC01 gene expression. Total RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and its integrity was examined by electrophoresis. The RNA was treated with DNAse I (Promega, USA) and cDNA was synthesized using the SuperScript™ III First-Strand Synthesis System for RT-qPCR (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) reactions were carried out using a Platinum Sybr Green Supermix (Invitrogen, Waltham, MA, USA) and run in an ABI 7500 qPCR thermocycler (Applied Biosystems, Waltham, MA, USA). Expression data were normalized using the 2-ΔCt method. The constitutive Elongation Factor-1 alpha (EF-1 alpha) housekeeping gene was used as internal control for tobacco (Schmidt & Delaney, 2010 (link)) and teak (Galeano et al., 2014 (link)). Primers were designed using the primer3 software (https://bioinfo.ut.ee/primer3-0.4.0/) (Table S1). We used three biological replicates and two technical repetitions for each transgenic line.