Nucleospin rna plant and fungi kit

Total RNA was extracted using the NucleoSpin^® RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany) according to the protocol of the manufacturer. The RNA integrity was measured via RNA electrophoresis, and the RNA concentration was quantified using a NanoDrop^TM One (Thermo Fisher Scientific, Waltham, MA, United States). The remaining DNA was removed by using the DNAase I RNase-free kit (Ambion, Applied Biosystems, Austin, TX, United States). Contamination with DNA was discarded in the sample sets by running a control PCR with aliquots of the same RNA that had been subjected to the DNase treatment but not to the reverse-transcription step, using an Eppendorf Mastercycler^® Personal (Eppendorf AG, Hamburg, Germany). The High-Capacity cDNA Reverse Transcription kit MultiScribe^TM (Applied Biosystems, Austin, TX, United States) was used to synthesize complementary DNA (cDNA) from 2 μg of total RNA. The synthesis was made with heat denaturation of the RNA according to the instructions of the manufacturer.

Gene expression analysis of the potato plantlets was performed using the quantitative RT-PCR microplate/DNA chip low density (qPFD^®) method [12 ,35 (link)]. Briefly, RNA was extracted using a Macherey Nagel Nucleospin^® RNA plant and fungi kit, and DNase treatment was performed during extraction according to the supplier’s instructions.
RNA quantification and quality control were performed for each sample. The yield and quality of the extracted RNA were assessed with a spectrophotometer (Nanodrop ND-1000). For each RNA sample, a reverse transcription (RT) step was performed from RNA normalized to 200 ng/μL with an Invitrogen™ SuperScript™ II reverse transcriptase kit according to the supplier’s instructions. The expression levels of 28 defense genes (Table 4) were quantified in triplicate by RT-PCR (SYBR Green) with the qPFD^® tool [12 ]. Results were analyzed according to the 2-ΔΔCT method, which provides the relative expressions of defense genes in a given sample compared with a control sample (water), expressions normalized by the geometric mean of 3 reference genes (i.e., tuA, actin, and GAPDH) [12 ] from that sample and transformed into log 2 (gene expression profiles are visualized as a density map called a heat map). Overexpression and repression levels of the defense genes were considered significant above a threshold ≥2 or ≤2, respectively.

RNA was isolated from dry seeds (0 HAI) as well as from the developing embryos/seedlings at 4, 12, and 24 HAI. The embryos and seedlings were isolated from the endosperm using tweezers, then frozen in liquid nitrogen and stored at −80 °C. For each extraction, approx. 100 embryos and seedlings that were ground in liquid nitrogen and a NucleoSpin RNA Plant and Fungi Kit (Macherey-Nagel, Duren, Germany) were used to isolate the total RNA. The RNA was purified using Qiagen RNase-Free DNase set (Qiagen, Venlo, Netherlands) and an RNeasy MinElute Cleanup Kit (Qiagen, Venlo, Netherlands). The concentration and quality of the isolated RNA was evaluated using an ND-1000 NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was produced using a Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). The primers that were relevant to the genes that were studied are listed in Table 2. The real-time PCR as well as the calculation of the relative expression level were done according to Betekhtin et al. [47 (link)].

RNA extraction was performed using the NucleoSpin RNA Plant and Fungi kit (Macherey‐Nagel GmbH & Co. KG, Düren, Germany) according to the manufacture’s recommendation. The RNA quality was assessed using a Qubit (Thermo Fisher Scientific, Waltham, USA) and an Agilent Bioanalyzer 2100 (Agilent Technologies, CA, USA). The NEB Next Ultra RNA Library Prep (NEB, CA, USA) kit based on the polyA method was used for RNA library preparation. Samples were sequenced on a NovaSeq 6000 system (Illumina Inc., CA, USA) and 150 bp paired-end reads were generated. Library preparation and sequencing were performed by Novogene Co., Ltd, Beijing, China.

Reverse transcription quantitative real-time PCR (RT-qPCR) was performed to analyze miraculin expression according to abiotic stress treatment and period of bioreactor culture in transgenic callus. TC3 (100 mg) cryopreserved in liquid nitrogen was used to extract total RNA and pulverized with Tissue-Lyser II (QIAGEN, Germany). Total RNA was extracted from the transgenic callus following the protocol of the NucleoSpin^® RNA Plant and Fungi kit (MACHEREY–NAGEL GmbH & Co., Germany). The extracted total RNA was used for cDNA synthesis using ReverTra Ace^® qPCR Master Mix (TOYOBO, Japan), which was then diluted to a concentration of 100 ng μL⁻¹. For RT-qPCR, we mixed a 20 μL reaction containing 10 μL TB Green Premix Ex Taq (Takara, Japan), 0.5 μL of each forward primer and reverse primer, 7 μL sterile distilled water, and 2 μL of cDNA and performed miraculin gene expression analysis using the CFX96 Touch™ Real-Time PCR System (Bio-Rad, California). The miraculin gene was amplified using primers miraculin-RT Fw (5′-CATCAATTTCTCGGCGTTCAT-3′) and miraculin-RT Rv (5′-AAACCACTACCACAAAACTCCT-3′). As a reference, the DcActin gene was used for normalization.

For the 486–1 isolate, total RNAs were extracted from leaf material using the Nucleospin RNA plant and fungi kit (Macherey Nagel). The GVT genome was completely sequenced by Sanger sequencing of cDNA clones of overlapping PCR products, including for the 3’ terminal sequence, for which a polyA-anchored PCR was used. The 5’ terminal sequence was determined 5’ RACE (Invitrogen), following the instructions of the supplier. The cDNAs were dC- and dG-tailing in separate reactions, and amplified by PCR using a GVT-specific internal primer and oligo-dG or oligo-dC primers, respectively. At least four clones from each reaction were sequenced. The obtained 5’end sequences were all identical.

For reverse transcription-quantitative real-time PCR (RT-qPCR) analysis, total RNA was extracted from frozen leaves and roots using the NucleoSpin RNA plant and Fungi kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions. RNA concentration and purity were quantified using a NanoDrop ND-1000 photospectrometer (Thermo Scientific, Germany). RNA integrity was confirmed on a 1.2% agarose gel. DNase I treatment was conducted on 2 μg RNA. Complementary DNA (cDNA) was synthesized using the RevertAid H Minus First Strand kit (Thermo Scientific) with oligo-(dT)₁₈ primers. After elution in 20 μl RNase free water samples were subsequently diluted by adding 20 μl deionized water to the final volume of 40 μl. RT-qPCR reactions were performed in a total reaction volume of 5 μl containing 2.5 μl Power SYBR Green Master Mix (Thermo Scientific), 0.5 μM forward primer, 0.5 μM reverse primer, and 0.5 μl of cDNA. ACTIN2 was used as a reference gene. The thermal profile used for all RT-qPCRs was: 2 min 95°C; 40 cycles of (3 s 95°C; 30 s 60°C); the data were analyzed by the 2^−ΔΔCt method.^{28 (link)} Specific Primers for the genes involved in nitrogen assimilation and NO scavenging were designed using Quantprime software.^{29 (link)} All sequences of gene-specific primers are provided in Supplementary Table S1.