Quantitative Analysis of Transcript Levels

Specific transcript levels were determined using quantitative reverse transcription-PCR (qRT-PCR). Total RNA was isolated from log phase cultures using RNeasy Mini kit (Qiagen). RNA was treated with DNase1 on column using an RNase-Free DNase Set (Qiagen) and cDNA was synthesized using an iScript cDNA synthesis kit (BioRad). qPCR was performed using diluted cDNAs and Power SYBR green PCR Master Mix (Life Technologies) with oligonucleotide pairs specifically targeting transcripts for wild-type E6 and RNAi^R HA:E6 and HA:E6ΔG. The positions of these primers are described in Tiengwe et al. (2017) (link). TbZFP3 (Tb927.3.720, nts 241–301) was used as the control amplicon. Amplification was performed using an Applied Biosystems StepOne real-time PCR system (Life Technologies). For each transcript, postamplification melting curves indicated a single dominant product. All calculations and normalizations were done using StepOne software, version 2.2.2. Reactions were performed in triplicate, and means ± standard deviation (SD) for three biological replicates are presented.