Qualified rnase free dnase

To verify the presence of the transgene in tobacco shoots or plants, total RNA was extracted from 50 mg of fresh tissue using Genelute Mammalian Total RNA kit (Sigma-Aldrich). To avoid the presence of any residual DNA, each RNA sample was treated with five units of Qualified RNAse-free DNAse (Promega) for 30 min at 37°C. The cDNA was synthesized using 2 µg of total RNA with RevertAid M-MuLV Reverse Transcriptase (Fermentas Int.). All PCR reactions were carried out using the ribosomal RNA QuantumRNA 18S Internal Standards (Ambi-on) as internal control. The primers and the conditions used for the RT-PCR reactions were identical to those described for the analysis of DHR expression in the insect.

500 ng of extracted RNA was treated with Qualified RNAse-free DNAse (Promega) for 30 min at 37°C and reverse-transcribed by using the QuantiTect Reverse Transcription kit (Quiagen). To minimize variations during the cDNA synthesis step, all RNA samples were reverse-transcribed simultaneously. The sequences of the primers used for the amplification of the
SpodopteraDHR genefragments were Fw:5'-GACGGCGTGTGGCACAACTAC-3' and Rv: 5'-CGGCGAGGCTGAGCGAGTAG-3'. The size of the fragment obtained was 117 bp. Real-time PCR reactions were performed in triplicate in 20 µL reaction volumes, and the general protocol described in
Conte et al. (2010)
was followed. For each transcript, the qRT-PCR values obtained, recorded as threshold cycle numbers, were analyzed by means of the ABI Prism 7900HT Fast Sequence Detection System software (Applied Biosystem), normalized against an internal control (β-actin), and then expressed as percentage values to control.