480 real time pcr machine

Total RNAs were isolated from the basal stem, leaf, leaf sheath, root, stem, and spike of wild-type plants at different growth stages, including seedling, tillering, stem elongation, booting, and heading, using a centrifugal filter kit according to the manufacturer’s instructions (Takara). First-strand cDNA synthesis was performed with 1 μg of total RNA using the PrimeScipt RT reagent kit with gDNA Eraser (Takara), according to the user manual. cDNA samples were diluted 3-fold, with 1 μL used for further analysis. qPCR analyses were performed using gene-specific primers Q-HNT1-F and Q-HNT1-R (Supplemental Table S1) in the reaction system of SYBR Green Supermix (Bio-Rad) on a Roche 480 real-time PCR machine (Roche). The basal stem of hnt1 and wild-type seedlings in the tillering stage was also used to analyze the gene expression of auxin-related genes. The barley ACTIN gene was used as an internal control. RT-qPCR was carried out with three biological and technical replications. The 2^−ΔΔCq relative quantification method was used to evaluate quantitative variation. All primers are listed in Supplemental Table S1.

Total RNA was extracted using the EZ-press RNA Purification Kit (EZBioscience, USA) according to the manufacturer’s instructions. Real-time PCR was performed using the EZ-press One Step qRT-PCR Kit (EZBioscience) on the Roche 480 real-time PCR machine (Roche). β-Actin served as an internal control for real-time PCR. All primers used for real-time PCR are listed in Additional file 1: Table S1.

Procedures were described previously [25 (link),26 (link)]. Total RNA was isolated using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies/Applied Biosystems, Foster, CA, USA) according to the manufacturer’s instructions.10 ng cDNA was then used in qPCR using SYBR green kits (Thermo Fisher Scientific, Waltham, MA, USA) on a 480 real-time PCR machine (Roche Applied Science, Penzberg, Germany). The specific primers used in qPCR were as follows: primers for TXNDC9 (forward: 5′-CTGCTTCAGACTACCAAACTGG-3′, reverse: 5′-CTCTGTAGAAATGGCAAACCACA-3′) and GAPDH (forward: 5′-ATCAGCAATGCCTCCTGCAC-3′, reverse: 5′-CGTCAAAGGTGGAGGAGTGG-3′). RT-PCR results were calculated using the ∆-Ct method.