Spectrum plant total rna extraction kit

The putative transformants were screened by PCR for transgene integration using specific sets of primers (UGPase-S F & GBSS-Int R; Supplementary Table 2) to amplify 600 bp fragment encompassing UGPase-S and GBSS-Int. PCR amplification (Supplementary Table 3) was carried out in a thermal cycler (Eppendorf, Germany) programmed with a hot start at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1.5 min and a final extension at 72°C for 7 min. The amplified PCR products were resolved on 1% agarose gel, visualized on a UV- trans-illuminator, and photographed using a gel documentation system. Transgene expression in the PCR-confirmed transgenic lines was confirmed by RT-PCR. Total RNA was extracted from the UGPase RNAi potato lines and wild-type control potato plants using the Spectrum^TM Plant Total RNA Extraction kit (Sigma, USA) according to the manufacturer’s protocol. The cDNA was synthesized from 800 ng of total RNA using the PrimeScript^TM single-strand cDNA Synthesis Kit (Takara Bio Ink). RT-PCR was done using the same primer set used for PCR analysis. The amplified RT-PCR products were electrophoresed on 1% agarose gel, visualized on U.V.- transilluminator, and photographed using a gel documentation system.

For each collection points at 0, 5, 10, 15, 20 and 30% of weight loss, three biological replicates of 50 berries were randomly selected and immediately frozen in liquid nitrogen. Samples were stored at −80 °C until molecular analyses. Total RNA was extracted from deseeded berries using the Spectrum^TM Plant Total RNA extraction kit (Sigma-Aldrich) starting from 100 mg of material, and RNA quantity was checked using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). Total RNA was treated with DNase I (Invitrogen, Thermo Fisher Scientific) following the manufacturer’s instructions. For each biological replicate, first-strand cDNA was synthesized starting from 500 ng of total RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer’s instructions. Reactions were carried out using specific primers (Supplementary Table S1), and Power SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific) as reported in Gambino et al.^{74 (link)}. Three technical replicates were run for each biological replicate, and the geometric mean of the expression ratios of two housekeeping genes (ubiquitin and actin) were used as the normalization factor. The results were calculated as expression ratios (relative quantity, RQ) to Moscato bianco berries at P0, before dehydration.

The quantification of miRNA expression levels was carried out by RT-qPCR following the protocol by Shi and Chiang (2005) (link), with the modifications reported in Pantaleo et al. (2016) (link). For target quantification, total RNA was extracted using the Spectrum^TM Plant Total RNA extraction kit (Sigma-Aldrich, Inc.) and RT-qPCR assays were performed as reported by Pantaleo et al. (2016) (link). Expression levels of miRNAs and related target genes were quantified after normalization to either 5.8S rRNA and U6 or VvUBI and VvACT endogenous genes used as internal controls, respectively. Specific primer pairs used in RT-qPCR reactions are listed in Supplementary Table S1. Transcript and miRNA levels were expressed as the mean and standard errors were calculated over five biological replicates.