Microamp fast optical 96 well reaction plates with barcode

qRT-PCR was performed using TB Green™ Premix Ex Taq™ II (Takara, Dalian, China) on the StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Twenty microliter-reactions were performed in MicroAmp Fast Optical 96-Well reaction plates with barcodes (Applied Biosystems, Foster City, CA, USA) with each reaction containing 2 μl cDNA, 10 μl 2 × qPCR Mix, 0.4 μl 50 × ROX Reference Dye, 0.8 μl each of the forward and reverse primers, and 6 μl of nuclease-free water. The PCR program involved a two-step process of initial denaturation at 95 °C for 30 s followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s where the fluorescence signal was detected. Four technical replicates were included for each reaction. Melting curve data were gathered from 95 °C maintain 15 s down to 60 °C hold 1 min, then up to 95 °C keep 15 s (with increments of 0.3 °C), with fluorescence signals detected during this increase.

RT-qPCR reactions were performed using the Universal SYBR Green detection system (Invitrogen, Carlsbad, CA, USA). Amplifications were conducted using 0.1 mL MicroAmp Fast Optical 96-well reaction plates with barcodes (Applied Biosystems, Foster City, CA, USA), sealed with MicroAmp™ Optical Adhesive Film (Applied Biosystems). Gene expression was quantified by the ∆∆Ct method⁴⁴ . Reactions were performed on a StepOne™ 7500 fast Real-Time PCR System (Applied Biosystems) at the Plant-Pest Interaction Laboratory at the Universidade de Brasília (UnB), Brasília-DF, Brazil. The reaction mix consisted of iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) (1X), forward and reverse primers (0.2 µM each), 1:20 cDNA (2.0 µL) and adjustment with nuclease-free water to a final volume of 10 µL. Amplification conditions were as follows: initial hold at 52 °C for 2 min, denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Each gene was analyzed in three biological replicates, with technical triplicates included per repetition. Primer specificity was also double-checked by confirming the presence of a single peak on melting curves in RT-qPCR analysis (Supplementary Figure S1), together with agarose gel analysis of all amplicons obtained via RT-PCR. Supplementary Figure S2 shows an example of specificity for the tested gene UBI3.

RNA was isolated 48 h after transient transfection using RNeasy kit (QIAGEN, Hilden, Germany) following the company’s instructions. cDNA was synthesized using Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) and oligo-dT primers according to instructions. qPCR reactions were performed in MicroAmp^® Fast Optical 96-Well Reaction Plates with Barcode (Life Technologies, 4346906) in a StepOnePlus™ Real Time PCR-System (Applied Biosystems, Darmstadt). Two hundred nanograms of cDNA, 0.1 μl each of forward and reverse primers (20 μM), and 10 μl of SYBR Green mix (KAPA SYBR FAST ABI Prism, Peqlab, Erlangen, Germany) were combined in a final volume of 20 μl. Cycling conditions were 3 s 95°C, 30 s 60°C followed by a melting curve analysis of the PCR product. Data were analyzed with the StepOnePlus™ software. Data were normalized to beta-actin (primers 1760/1761). TBX3 expression was measured with primers 1924/1925, p21 expression with primers 1772/1773 (see Table S5 in Supplementary Material).