Sv rna isolation system kit

Total RNA was extracted from whole body of each individual with the SV RNA Isolation System kit (Promega), following the manufacturer’s instructions. RNA samples were quantified in the spectrophotometer Nanodrop 2000 (Thermo Fisher Scientific) and their integrity was verified by electrophoresis in agarose gel 1% (w/v). First-strand cDNA synthesis was made from 1 µg of total RNA using the Improm-II™ Reverse Transcription System kit (Promega).
Since there was no genome sequence available for M. interrupta, primers were initially designed to amplify and sequence partially the cDNA of mfe and jhe in this species, based on the coding sequences of orthologous genes from the closely related species M. quadrifasciata (Table S1). The OrthoDB : Database of Orthologous Groups program was employed to search for the mfe sequence in the genome of M. quadrifasciata, using mfe sequence of Apis mellifera as query (GenBank accession number: NM_001327966.1). The jhe sequence of M. quadrifasciata was obtained through a search directly in GenBank (accession number: EU099312.1). Sequencing was carried out on an ABI 3130 Genetic Analyzer (Applied Biosystems) with Big Dye^® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), following manufacturer’s instructions. The obtained sequences were compared to the sequences deposited in GenBank database using the Blast tool .

The RNA extraction was done according to the procedure described by Wang et al. (2015 (link)) in the manual of SV RNA Isolation System kit (Promega) and the centrifugation was used at 12,000–14,000 rpm. Yield of RNA obtained was determined by using a Spectrophotometer at 260 nm wavelength. The manual of Takara Prime Script 1st strand cDNA synthesis kit was consulted to synthesize the cDNA. The reaction tubes were incubated in heat-block at 70°C for 15 mins to inactivate the reverse transcriptase before doing the PCR and amplification. Aliquots of the single strand cDNA of the treated plants either at 0, 15, or 20 mM were subjected to quantitative RT-PCR analysis on the rbcL gene. The expression of the Co-GAPDH (glyceraldehydes-3-phosphate-dehydrogenase) gene was considered as the internal control. Using a Rotor-Gene 6000 Series Software 1.7 (Build 65), the expression data were evaluated.