Oligo dt adaptor primer

Total RNA was isolated using RNA isoreagent (TaKaRa Bio Inc., Shinga, Japan) according to the manufacturer's protocol. cDNA was synthesized from 1 μg total RNA in a 10 μL aliquot using Moloney Murine Leukemia Virus reverse transcriptase (M-MLV, TaKaRa Bio Inc.) at 42°C for 1 h with the oligo-dTadaptor primer (TaKaRa Bio Inc.) following the manufacturer's protocol. Real-time quantitative PCR was performed using 0.5 μL of reverse transcription production in a 25-μL reaction volume with SYBR Premix Ex Tag (TaKaRa Bio Inc.) on an ABIPRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using gene-specific primers: β-tubulin was used as a house-keeping gene according to Xu et al. (2010 (link)), and the GmNPR1 Gene-Bank accession number is FJ418595; upstream primer 5′-ATGGCAAGGTTGGATATGAAGC-3′ and downstream primer 5′-TGGCAGGTCTACACGCATCA-3′; the amplification size of the product was 132 bp. Each sample was run in triplicate. The relative transcript abundance was calculated using the 2^−ΔΔCT method.

Total RNA was extracted from the antennae of male moths at 1–5 days after eclosion using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), treated with DNase I, and reprecipitated. RNA was reverse transcribed using an oligo(dT) adaptor primer (Takara-Bio, Otsu, Japan) and AMV reverse transcriptase (Takara-Bio) at 42 °C for 35 min. The cDNA of BmOR1 and B. mori ribosomal protein49 (rp49) was amplified using Ex Taq DNA polymerase (Takara-Bio) and the primer pairs for BmOR1 (5′-CGGAAAAACAACTGAACGAA-3′ and 5′-CCGTTATGAAGCGACCAGTT-3′) and B. mori rp49 (5′-CAGGCGGTTCAAGGGTCAATAC-3′ and 5′-TGCTGGGCTCTTTCCACGA-3′), with thermal cycling at 95 °C for 2 min, followed by 30 cycles at 95 °C for 30 s, 50 °C for 15 s, and 72 °C for 20 s, and 72 °C for 5 min. Equal amounts of the PCR products were separated by electrophoresis on 2.0% agarose gels. No PCR products were produced when reverse transcriptase was excluded during reverse transcription, and sequence analysis confirmed the identity of the cDNA products. For confirmation of the deletion sequence in BmOR1-/BmOR1- male antennal transcripts, the following primer pair was used (sense: 5′-ACCAAGGTCAGTTTCGGCTATAAAAG-3′ and antisense: 5′-CACAAGTAACCAAATTCATCATCACAG-3′).

To assess the transcript level of EGFR, total cellular RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer protocol. The synthesis of the first strand cDNA was performed with Reverse Transcription System using 0.5 µg of RNA and Oligo dT-Adaptor Primer in a 10 μL reaction volume Mixture (TAKARA, Dalian, China). PCR was performed in a 40 μl reaction mixture consisting of 0.25 μl of TaKaRa Ex Taq HS (5 U/μl), 0.5 μL of 20 μM specific primers, 10 μl cDNA, and water as needed. Specific forward and reverse primers (Invitrogen) to produce for optimal amplification of reverse-transcribed cDNA for EGFR were as follows: 5′-CGTCCGCAAGTGTAAGAA-3′ and 5′-AGCAAAAACCCTGTGATT-3′, for β-actin were 5′-TGAGCGCGGCTACAGCTT-3′ and 5′-TCCTTAATGTCACGCACGATTT-3′. The β-actin gene was used as an endogenous control for normalization.