Onescript plus cdna synthesis kit

Tissue samples were shock-frozen in liquid nitrogen and total RNA was obtained using the FavorPrep Tissue Total RNA Kit (FATRK001, Favorgen, Taiwan) after tissue homogenization using lysing tubes (Lysing Matrix E, MP Biomedicals, Germany) on a Precellys 24 (Bertin Instruments, France). Reverse transcription was performed using the OneScript Plus cDNA synthesis kit (G236, ABM Good, Canada). For quantitative PCR BrightGreen Express 2× Mastermix (MasterMix-EL, ABM Good, Canada) was used. Exon spanning primers for DAO, HNMT and histidine decarboxylase (HDC) were designed using Primer3 software (Online Resource Table 1). The housekeeping gene RPLP0 was used for normalization [56 (link)].

RNA was extracted using the NucleoSpin RNA kit (Macherey and Nagel, Bethlehem, PA, USA) from three biological replicates, each made of 20 third instar larvae or 50 larval fat bodies. RNA concentration and quality were evaluated by measuring in 0.1 N NaOH the OD at 260 nm and the ratio at 260/280 nm, respectively, and by electrophoresis on 1.2% agarose gels. Reverse transcription of DNase-treated RNAs (1 μg) was carried out using the OneScript^® Plus cDNA Synthesis Kit (ABM Good, Richmond, BC, Canada) with the random primers provided in the kit. RT-qPCR was performed on a CFX Connect Real Time PCR system (Bio-Rad, Hercules, CA, USA) with a two-step reaction using SYBR green ExcelTaq™ Master Mix (SMOBIO, Hsinchu City, Taiwan) and the oligonucleotides reported below. The relative expression of each target gene was determined by the Pfaffl method using the α-tubulin as the normalizer. The fold induction resulting from the different pairs of samples was averaged and the p value was calculated using the Student’s t-test.
Hex-A_for CAATGTGCGGTACATCTGCG
Hex-A_rev TTGGGATGGAAGCGGTACAC
Hex-C_for GTCGCTTTTGCCTGGAAGTG
Hex-C_rev TGGTGACCTTTCAGCGAGAC
α-tubulin_ for TGTCGCGTGTGAAACACTTC
α-tubulin_ rev AGCAGGCGTTTCCAATCTG