Anchored oligo dt primers

RNA extracted from whole intestinal samples, was used to synthesise first strand cDNA using a VersoTM cDNA Kit (Thermo Scientific) and anchored oligo-dT primers (Thermo Scientific) according to the manufacturer's instructions. All qRT-PCR reactions were run on the Applied Biosystems StepOnePlus machine. The programming was carried out using the computer-based StepOnePlus software. Primers were designed using the Universal ProbeLibrary Assay Design Centre (Roche).

The total RNA for each pool of 10 insects per biological replicate was extracted after sample homogenization at room temperature with a plastic pestle using 500 µL of TRIzol reagent (Thermo Fisher scientific, MA) following the manufacturer’s instructions. Traces of genomic DNA were removed by DNase treatment using the TURBO DNA-free kit (Thermo Fisher scientific). The total RNA quantity and purity was evaluated on a Nanodrop Spectrophotometer (Thermo fisher Scientific), and the integrity of the RNA was visualized in 1.2% agarose gel electrophoresis. The synthesis of cDNA was performed using the Verso cDNA Synthesis kit (Thermo fisher Scientific). Each retro-transcription synthesis was conducted using five hundred ng of total RNA as template and adding anchored-Oligo (dT) primers, following manufacturer recommendations (Thermo Fisher scientific).

Total RNA was extracted from cells using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma, Saint-Louis, MO, USA) or the GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. RNA (125 ng to 400 ng) was reverse-transcribed using anchored oligo dT primers (Thermo Fisher Scientific, Waltham, MA, USA), and SuperScript™ II reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) according to vendor’s instructions. cDNAs were analyzed in triplicate by quantitative real-time PCR using 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) or QuantiTect SYBR^® Green Master Mix (Qiagen, Hilden, Germany). The oligonucleotides used (T4Oligo, Irapuato, México) are listed in Table 1. Gene expression was normalized by using RPL32 as housekeeping gene.

The reverse transcription reactions were performed in a total volume of 20 μl using 0.5–2 μg of total RNA, 500 ng/μl Anchored Oligo-dT primers (Thermo Fisher Scientific), and 200 U Superscript III™ reverse transcriptase (Invitrogen) following manufacturer’s first-strand cDNA synthesis protocol. All cDNA samples were diluted in 9 volumes of sterile de-ionised water and stored at −20 °C until RT-qPCR analyses. Semi-quantitative RT-PCR reactions were carried out using PlatinumTaq (Invitrogen) in a C1000™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) for 35 cycles (30 s. denaturation at 94 °C followed by 30 s. annealing at 60 °C and 1 min extension at 72 °C). Quantitative PCR was performed with an iCycler iQ™5 Real-Time Detection System (Bio-Rad Laboratories) using 8 μl diluted cDNA, 0.4 μM of the forward (1 μl) and reverse (1 μl) primer, and 10 μl 2× SYBR-Green I dye master mix (QuantiTect^® SYBR^® Green PCR kit, Qiagen) in a reaction volume of 20 μl. The RT-qPCR reaction mix was denatured at 95 °C for 10 min and then subjected to 45 amplification cycles (10 s. denaturation at 95 °C, 45 s. annealing at 60 °C and 30 s. extension at 72 °C) [3 (link)]. Under these optimized qPCR conditions, each primer set presented amplification efficiency close to 100% (Fig. S4) and a single peak in melt-curve analyses.