Reverse primer

The 3′UTR fragment of GSTM3 was amplified and cloned downstream of a luciferase reporter system in the pISO vector (Addgene plasmid #12178). Amplification of the 3′UTR fragment of GSTM3 was performed with the following primers (Sigma-Aldrich):
5′-TTACAGAGCTCATCCTGTCCGTAAGGGGTCA-3′ (forward),
5′-TGTAATCTAGAAGTCTGAAATACTGCCTTTATCAC-3′ (reverse).
To generate part or full mutation of the binding site for hsa-miR-200c-3p at the 3′UTR of GSTM3, the reverse primers (Sigma-Aldrich) listed below that contained nucleotide mismatches were used:
Mut 1     5′-TGTAATCTAGAAGTCTGAAACACTGCCTTTATCAC-3′
Mut 2     5′-TGTAATCTAGAAGTCTGAAATACATCCTTTATCAC-3′
Full mutation   5′-TGTAATCTAGAAGTCTGATCACAATCCTTTATCAC-3′
Putative miRNA-mRNA seed-site interactions for hsa-miR-200c-3p were analyzed in silico using TargetScan [39 (link),40 (link),41 (link)]. Sequences of 3′UTRs and the predicted site types of the different GSTs were also adapted from TargetScan.

Thirty nine unrelated individuals from the 3 populations were characterised by the Y-chromosome karyotype and used in the study. Five microsatellite markers capable of amplification from An. culicifacies sp. A and sp. B from India [16 (link)] were selected for this study viz. AcAIIB5, ACAVB93, AcAVIB213, AcA 36 and AcA59. Fluorescent-labelled (6-FAM®, NED®, VIC® and PET®) forward primers (Applied Biosystems, UK) and non-labelled reverse primers (Sigma-Aldrich) were used in a multiplex PCR reaction. Each individual reaction of 5 μl consisted of 0.5 μl of the 10x primer mix (each primer at 2 μM), 2.5 μl of Type-it Multiplex PCR Master Mix (QIAGEN), 1 μl of MQ water and 1 μl of genomic DNA (10–20 ng). The amplification conditions were; initial denaturation at 95 °C for 5 min, followed by 26 cycles of 95 °C for 30 s, 57 °C for 120 s, and 72 °C for 30 s. This was followed by final extension at 60 °C for 30 min. The PCR product was diluted with 5 μl MQ water and 0.5 μl of this was mixed with 9.5 μl of a mix consisting Hi-Di Formamide® (Applied Biosystem) and GeneScan – 500 LIZ Size Standard (37:1) prior to genotyping on an ABI 3730 automatic DNA sequencer using GeneMapper® v.3.7software (Applied Biosystems, USA).

RNA extraction was performed on pre-lysed samples stored at −80 °C, using the ReliaPrep RNA Cell Miniprep System (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The resulting RNA was measured using a Nanodrop 100 V3.8.1 (ThermoFisher Scientific, Waltham, MA, USA), and then it was stored at −80 °C. RNA was then reverse transcribed using a TaqMan Kit (Applied Biosystems, Waltham, MA, USA) with either 20 µL or 40 µL total reaction volume. The cDNA was then generated using a Verriti 96-well Thermal Cycler (ThermoFisher Scientific) using the following protocol: 10 min at 25 °C, 30 min at 48 °C then 5 min at 95 °C. The cDNA samples were stored at −20 °C, then analyzed by qPCR, with a GoTaq qPCR Master Mix (Promega). Master mix: 10 µL Power Sybr Green MM, 0.75 µL of 5 µM forward primer, 0.75 µL of 5 µM reverse Primer (Sigma, 100 µM stock), and 7.5 µL nuclease free water per well. 18 µL of the master mix was combined with 2 µL of cDNA, and qPCR was performed in a 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA), then it was analyzed using Applied Biosystems 7500 System SDS Software v2.0.5. A list of primers is provided in Table 2.