We further evaluated whether our RNA-Seq analysis results could be independently replicated using the quantitative reverse transcription polymerase chain reaction (RT-qPCR) technique as an independent tool. We randomly selected DE larval transcripts in our RNA-seq library and compared their relative fold changes to those we obtained for the same transcripts through RT-qPCR. Briefly, we generated five independent biological replicates (exposed and control) of the larvae, extracted and cleaned the respective total RNA libraries from our control and exposed larvae ab initio. We used the same methods and procedures we used to prepare the biological samples for the RNA-seq component of our study for sample preparations and total RNA extraction. We then reverse transcribed 1 μg of the total RNA using the iScript™ cDNA synthesis kit (BIO-RAD, Hercules, USA) on the Arktik thermal cycler (Thermo Scientific, USA), according to the manufacturer’s protocol.

We randomly selected eight DE transcripts for validation of differential expression. These transcripts were significantly induced or suppressed in the exposed larvae library relative to the control library to encompass and validate differential expression in both directions (up- and downregulated genes) of the libraries. We also selected four transcripts that were neutral (neither induced nor suppressed in the exposed library relative to the control library) as potential internal reference neutral/loading controls [41]. These reference transcripts consisted of CLIP-domain serine protease, glyceraldehyde 3-phosphate dehydrogenase (gapdh) and two uncharacterized genes (Additional file 1: Table S1) from VectorBase [28]. We ensured that all these transcripts were abundantly expressed in the RNA-seq libraries (based on their RPKM values) to ensure that their expression levels would be within the sensitivity of our Stratagene MX3005P RT-qPCR machine (Agilent Technologies, CA, USA). We obtained DNA sequences of respective genes from VectorBase [28] using the respective gene IDs (Additional file 1: Table S1) and designed primers (Additional file 1: Table S1) from these sequences in silico using primer3 software [42]. In all cases, we ensured that the melting (Tm) and annealing temperatures of the respective forward and reverse primers generated were similar, as determined by pDRAW32 version 1.1.142 software (http://www.acaclone.com) (Additional file 1: Table S1).

We first interrogated reference transcripts for their stable expression by performing RT-qPCR in three technical replicates for each of the five biological replicates on our Strategene MX3005P RT-qPCR machine (Agilent Technologies, CA, USA) using Fast SYBR Green I Master Mix (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions. We performed the PCR in reaction volumes of 10 μl for each replicate consisting of 1 μg cDNA template in three independent replicates with 5 μl of Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) in the presence of 0.4 picomoles of specific primers for the respective candidate reference transcripts. We carried out the reactions in a RT-qPCR thermal cycler (Stratagene MX3005P, Agilent Technologies, CA, USA) according to the manufacturer’s instructions. We involved thermo-cycling conditions that included an initial step of 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 55.0–63.2 °C (Additional file 1: Table S1) for 45 s and 72 °C for 1 min, followed by one cycle of 95 °C for 1 min, 55 °C for 30 s and 95 °C for 30 s for all the genes. We then assessed stability (non-differential expression) of these reference transcripts using BestKeeper software [43]. From this assessment, we identified gapdh and CLIP-domain serine protease transcripts as less variable [with a standard deviation of crossing point (CP) of 0.56 and 0.65, respectively] among the reference transcript candidates. We thus adopted these two genes as our internal housekeeping transcripts for assessment of expression of the eight randomly selected transcripts. We then separately performed the RT-qPCR for each of these transcripts under similar reaction and thermocycling conditions as had been previously employed in the assessment for stable expression of the reference transcripts above, but with gapdh and CLIP-domain serine protease as internal reference/loading controls.

We computed and compared relative expression (means, fold changes and p-value) of the eight transcripts using Relative Expression Software Tool (REST)-384 version 2 software [44]. We then used these data to evaluate validity of the transcriptomes by comparing the fold changes obtained here (RT-qPCR) to those obtained earlier (RNA-Seq) for the eight genes through Pearson correlation analysis.

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