Rnaaqueous micro total rna isolation kit

Total RNA was extracted from all microdissected samples using RNAaqueous Micro Total RNA Isolation Kit (Thermo Fischer Scientific, MA, USA), according to manufacturer’s instructions. In order to compare the amount of RNA recovered per sample, all extracted RNA samples were eluted in the same volume (15 µL) of water. The procedure was done in RNase free environment using RNAseZAP (Ambion, MA, USA), under ice. After extraction, the total RNA concentration was determined using a NanoDrop^® spectrophotometer (Thermo Fischer Scientific, MA, USA), at optical density 260 nm. Sample purity was checked by reading the plate between 230/260 nm and 260/280 nm. After quantification, total RNA was frozen at −80 °C until processing for transcription into cDNA.

The PicoPure RNA Isolation Kit (Life Technologies, MERK SA, Darmstadt, Germany) was used to extract RNA from whole biopsy samples, whereas for the microdissected samples, a specific kit for microdissected samples, RNAaqueous Micro Total RNA Isolation Kit (ThermoFischer Scientific, Waltham, MA, United States) was used. The extracted total RNA was quantified by spectrophotometry using a NanoDrop One Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific, Waltham, MA, United States) at an absorbance of 260 nm and frozen at −80°C.

Total RNA was extracted from the leaves of both plant varieties using RNAaqueous-Micro Total RNA isolation kit (Thermo Scientific, United States). NanoDrop One UV–Vis Spectrophotometer was used to check the concentration and purity of RNA (Thermo Scientific, United States). One microgram of total RNA was treated with DNAse I and the treated sample was used to synthesize cDNA with Revert PrimeScript cDNA synthesis kit (Takara Bio, Japan). The expression of mannitol-induced stress-responsive transcripts was analyzed by qRT-PCR using Applied Biosystems 7,500 Fast Real-Time PCR System. The following program was used: one cycle of 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 30 s. The qRT-PCR reactions (10 μl) included 30 ng of cDNA in 1 μl, TB Green Premix Ex Taq II (TliRNaseH Plus) (1×), PCR Forward Primer (10 μM) 1 μl, PCR Reverse Primer (10 μM) 1 μl, ROX Reference Dye (50×) 0.2 μl, and sterile purified water 1.8 μl.