Sensifast syber lo rox mix

Two micrograms of RNA from different tissues (bulb, root, stem, leaf, and flower) of triplicate plants of N. pseudonarcissus ‘King Alfred’ were reverse transcribed to form cDNA using oligo dT primers and the Omniscript Reverse Transcription Kit (Qiagen) according to given manufacturer’s protocol. Gene specific primers were designed using Integrated DNA Technology (www.idtdna.com) and Tm Calculator, New England Biolabs (tmcalculator.neb.com), to select suitable annealing temperatures. The sequences for all of the primers used in this study are listed in Supplementary file 4. SensiFAST SYBER Lo-ROX mix (Bioline) was used to prepare a 20 μl reaction containing 1x SensiFAST SYBER Lo-ROX mix, 200 μM of each forward and reverse primer and 3 µl of cDNA sample. Each experiment was performed in triplicate and histone was used as an internal reference gene. Real-time quantitative PCR was performed on CFX Connect Real-Time PCR System (BioRad). PCR conditions for amplification were 95 °C for 3 min, 95 °C for 10 sec and annealing temperature (varies with gene primers, see Supplementary file 4) for 30 sec for 40 cycles. This was followed by a dissociation step (as provided by software) −95 °C for 10 sec, 65 °C for 5 sec and 95 °C for 5 sec. The comparative 2^−ΔΔCt method^{72 (link)} was used for relative quantification of the gene expression levels.

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Two micrograms of total RNA were used in a 20 μL reverse transcription reaction using High-Capacity cDNA Reverse Transcription Kits (AB, USC) for cDNA synthesis. For real-time PCR, QuantStudio 3 Real-Time PCR system (Thermo) was used. The real-time PCR reactions were performed in 20 μL volume containing 1 μL cDNA, 10 μL SensiFAST SYBER Lo-ROX Mix (BIOLINE), and primer pairs. The following primer pairs were used: C1GALT1, CDK1, K1F20A, F1F2C, BUB1, GBP1, PRF1, OAS2, BST2, and GAPDH (Supplementary Table S1).