Perfecta sybr green fastmix for iq

RNA was extracted using the Quick-RNA MiniPrep kit (Cat # R1054, Zymo Research, Irvine, CA, USA), normalized, and cDNA was synthesized with the Quanta Q-Script MiniPrep Kit (Cat # 101414-106, Quanta Biosciences, Beverly, MA, USA). Reactions were optimized for a 60 °C annealing temperature and run in duplicate on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using 25 µL volumes and the PerfeCTa SYBR Green FastMix for IQ (Cat # 101414-254, Quanta Biosciences). Primer sequences are summarized in Supplementary Table S2. Gene expression was normalized to a housekeeping gene, either β-actin or RPL32. Statistical analysis was performed using Δ-Ct values of the target and housekeeping genes while results are presented as fold changes normalized to control groups.

Barley leaves were pulverized in liquid nitrogen and total RNA extracted using Trizol-like reagent (Caldo et al., 2004 (link)). Genomic DNA was degraded by RNase-free DNase I (Ambion, Austin, TX, U.S.A.). SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used to synthesize first strand cDNA using 2 μg total RNA and oligo(dT)₂₀ primer. This cDNA was used as a template for qRT-PCR to determine expression of various target genes to barley Actin. The qRT-PCR was performed using a Bio-Rad iCycler (Bio-Rad, Hercules, CA, USA). Conditions for 20 μL reactions using PerfeCTa® SYBR® Green FastMix® for iQ (Quanta Biosciences, Gaithersburg, MD) were 95°C for 3 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min, then a melt curve was determined by starting at 55°C for 10 s and then increasing by 0.5°C every 10 s for 80 cycles. Three technical replicates for each biological sample in addition to four or five biological samples per treatment were included in each experiment. Target gene expression was calculated using the 2^−ΔCT method for the BSMV:target gene and BSMV:00-treated plants. The fold change due to silencing was calculated by dividing the expression value for each BSMV:target gene treated leaves by the mean value measured in BSMV:00 treated plants (Schmittgen and Livak, 2008 (link)).

E13.5 MEPMs were serum starved overnight in 0.1% FBS and then treated with desired growth factors and/or inhibitors as for RNA-seq. Total RNA was collected with the RNeasy Mini Kit (Qiagen Inc.). First-strand cDNA was then synthesized using a ratio of 2:1 random primers: Oligo(dT) with SuperScript II RT (Invitrogen). qPCR was performed using a Bio-Rad iQ5 Multicolor Real-Time PCR Detection System and analyzed with iQ5 Optical System Software (version 2.0; Bio-Rad, Hercules, CA). Reactions were performed with PerfeCTa SYBR Green FastMix for iQ (Quanta Biosciences Inc., Gaithersburg, MD) using 10 μM primers (Integrated DNA Technologies Inc., Coralville, IA). A list of qPCR primers used is available in Supplementary File 5. The following cycling protocol was used: step 1: 95°C for 3 min; step 2: 95°C for 10 s; step 3: 60° for 40 s; repeat to step 2 39× (total of 40 cycles), and a melting curve analysis was performed. In addition, PCR products were run on a 1.0% agarose gel to ensure correct amplicon size. β2m was used as an endogenous control.

RNA was extracted from inflorescences using TRIzol (Life Technologies) and treated with DNase while on an E.Z.N.A. Plant RNA spin column (Omega Bio-Tek). First-strand cDNA synthesis was performed using either qScript cDNA Supermix kit (Quanta BioSciences) or iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR reactions were performed on a Bio-Rad CFX Connect using either PerfeCTa SYBR Green FastMix for iQ (Quanta BioSciences) or iQ SYBR Green Supermix (Bio-Rad). Data analyses were carried out using the 2^− ΔΔCt method (Livak and Schmittgen 2001 (link)). Normalization was performed using AT5G15710 as a reference gene (Czechowski et al. 2005 (link)). Three biological replicates were used in each experiment.

35S:AlcR/AlcA:AIL6-amiR ant-4 and 35S:ALCR/AlcA:ANT-IR plants were mock treated or treated with ethanol vapor by placing 2 ml of water or 2 ml of 100% ethanol in 2 ml centrifuge tubes in half of the pots in a tray. The tray was covered with a plastic dome. Inflorescences were collected at the end of an 8 h (35S:AlcR/AlcA:AIL6-amiR ant-4) or 24 h (35S:ALCR/AlcA:ANT-IR) treatment. RNA was isolated using an RNeasy Plant Mini Kit (Qiagen) or TRIzol (Life Technologies). Samples isolated with TRIzol were further purified on an RNeasy column (Qiagen) and treated with DNase while on the column. First-strand cDNA synthesis was performed using Quanta qScript cDNA SuperMix (Quanta BioSciences) following the manufacturer’s instructions. Quantitative PCR (qPCR) was performed on a BioRad CFX96 or CFX Connect real-time PCR system using PerfeCTa SYBR Green FastMix for iQ (Quanta BioSciences) and primers listed in Supplementary Table S1. Data analyses were carried out as described previously (Krizek and Eaddy, 2012 (link)). At least two biological replicates were analyzed for each experiment.