Reveraid reverse transcriptase

To examine virus, total RNAs were isolated from young leaves excised from greenhouse shoots according to Meisel et al. (2005) (link). cDNAs were generated from 5 μg of the total RNAs using cloned ReverAid Reverse Transcriptase (Fermentas, United States) and were subjected to PCR. The primers used for amplification of a 245-bp fragment of 3′UTR were 5′-gtc-tct-tgc-aca-aga-act-ata-acc-3′ and gta-gtg-gtc-tcg-gta-tct-atc-ata-3′. The primers used for amplification of a 442-bp fragment of HC-Pro gene were 5′-cca-gga-atg-agc-gga-ttt-gtg-gt-3′ and 5′-cat-gtg-aaa-att-gtg-gat-agt-tat-cca-tca-c-3′. The primers used for amplification of a 990-bp fragment of RNA-dependent RNA polymerase gene were 5′-gaa-gga-aat-ttg-aaa-gca-gtt-ggagc-3′ and 5′-cat-tca-cra-art-acc-grc-aaa-tgc-a-3′. PCR products were separated by electrophoresis on 1.2% (w/v) agarose-ethidium bromide gels.

Whole brains, CC/CAs and VNCs from three stages of T. molitor were dissected in physiological saline (PS) (274 mM NaCl, 19 mM KCl, 9 mM CaCl₂, 5 mM glucose, and 5 mM HEPES, pH 7.0, Sigma-Aldrich, Poznań, Poland). Total RNA was extracted from ten samples of each tissue (10× brains, 10× CC/CAs and 10× VNCs) using the Insect RNA MicroPrep™Kit (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer’s protocols. The protocol included the in-column DNase I treatment to remove traces of gDNA and prevent the contamination of the RNA samples. ReverAid™ Reverse Transcriptase (Fermentas, Waltham, MA, USA) was then used to generate cDNA for PCR according to the manufacturer’s protocols.
A Bio-Rad T100 thermocycler (Bio-Rad, Hercules, CA, USA) was employed for PCR using the following cycling conditions: 95 °C for 3 min, followed by 34 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 60 s. The PCR products were separated using 2% agarose gels in TEA buffer and visualized using ethidium bromide. Gel products were purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corp., Irvine, CA, USA) and sequenced with BigDye Terminator v3.1 in an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocols.