Abi prism 7500 fast realtime platform

Tissue samples from selected homozygous T₄ lines were snap-frozen in liquid nitrogen and stored at −80 °C until further analysis. Total RNA was extracted using the RNeasy Plant MiniKit (Qiagen, Valencia, CA, USA), as per the manufacturer’s instructions. Two micrograms of DNA-free total RNA were used for cDNA preparation. Reverse transcription reactions were performed using the Transcriptor First-Strand cDNA Synthesis Kit RT-PCR (Roche, USA) according to the manufacturer’s instructions. Primers used in the expression study are noted in Supplementary Table S2. Quantitative real-time PCR analysis was performed by following SYBR Green (QuantiFast^TM SYBR Green PCR kit, QIAGEN) chemistry using the ABI PRISM 7500 Fast Realtime Platform (Applied Biosystems). Target genes were amplified by a two-step PCR reaction with an initial denaturation at 95 °C for 5min followed by 40 cycles of 95 °C for 30s and annealing/extension at 60 °C for 30s. Relative quantification for fold-changes were calculated and Ct values were normalized against wheat ARF (ADP-ribosylation factor, AB050957.1) as defined previously by Bhati et al. (2014) (link) and Alok et al. (2015) using the 2^–ΔΔCT method (Livak and Schmitteng, 2001 (link)). The specificity of the amplification was also assessed for each gene by dissociation curve analysis. A unique peak on the dissociation curve was confirmed for each gene.

For the confirmation of gene silencing, SYBR Green (Quanti-Tect SYBR Green RT-PCR Master mix, QIAGEN) based reactions were performed on ABI PRISM 7500 Fast Real-Time Platform (Applied Biosystems). The silencing of the gene was confirmed in the immature seeds of the developing grains at T₄. During the wheat transformation wheat lines derived from callus showing no integration of the vector were subsequently propagated along with the transgenic wheat as a control plants for comparative analysis. The main individual spikes of selected T₃ plants were tagged at the first DAA and tissue samples were harvested at 14 DAA in liquid nitrogen. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, United States), following manufacturer’s instructions. gDNA free cDNA was prepared using 2 μg of RNA. Transcriptor First Strand cDNA Synthesis Kit RT-PCR (Roche, Indianapolis, IN, United States) was used for cDNA preparation following the manufacturer’s guidelines. Further, level of silencing was checked using real time PCR primers targeting TaIPK1 CDS region other than RNAi fragments. The sequences of primers are listed in Supplementary Table S1. Relative expression level was quantified using 2^-ΔΔCt method after normalizing C_t values against wheat ADP-ribosylation factor 1 (ARF1) and 18S rRNA genes as an internal control (Livak and Schmittgen, 2001 (link)).