The expression profile of six of the most over-expressed detoxification genes previously associated with metabolic resistance in VK through microarray analysis (Kwiatkowska et al, 2013 (link)) were further assessed by qRT-PCR to validate their differential expression profile between mated and unmated mosquitoes (genes names and primer sequences are given in table S1 ). One microgram of total RNA from each of the three biological replicates for mated and unmated mosquitoes was used as template for cDNA synthesis using Superscript III (Invitrogen) with oligo-dT20 and RNase H (New England Biolabs), according to the manufacturer’s instructions. A serial dilution of cDNA was used to establish standard curves for each gene in order to assess PCR efficiency and quantitative differences between samples. The qPCR amplification was performed using a MX 3005 real-time PCR system (Agilent, Santa Clara, CA, USA) with Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent, Santa Clara, CA, USA) as described previously (Kwiatkowska et al, 2013 (link)). The relative expression and fold-change of each target gene in mated relative to unmated was calculated according to the 2−ΔΔCT method incorporating PCR efficiency (Schmittgen and Livak, 2008 (link)) after normalization with the housekeeping genes rsp7, encoding ribosomal protein S7 (AGAP010592) and Elongation Factor gene (AGAP005128).
Oligo dt20
Manufactured by New England Biolabs
Sourced in United Kingdom, United States
Oligo-dT20 is a synthetic oligonucleotide composed of 20 deoxythymidine (dT) nucleotides. It can be used to selectively isolate and amplify polyadenylated RNA molecules from total RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Mx 3005 real time pcr system, by Agilent Technologies (2 mentions)
Brilliant 3 ultra fast sybr green qpcr master mix, by Agilent Technologies (2 mentions)
Rnase h, by New England Biolabs (2 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Dntp mix, by New England Biolabs (1 mentions)
7 deaza dgtp, by New England Biolabs (1 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Stratagene mx3005p real time pcr system, by Agilent Technologies (1 mentions)
4 protocols using oligo dt20
1
Validation of Detoxification Gene Expression
2
Validation of Detoxification Genes
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The expression profile of six of the most overexpressed detoxification genes previously associated with metabolic resistance in VK through microarray analysis (Kwiatkowska et al., 2013 (link)) was further assessed by qRT-PCR to validate their differential expression profile between mated and unmated mosquitoes (genes names and primer sequences are given in Supplementary Table S1 ). One microgram of total RNA from each of the three biological replicates for mated and unmated mosquitoes was used as a template for cDNA synthesis using Superscript III (Invitrogen, Loughborough, UK) with oligo-dT20 and RNase H (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's instructions. A serial dilution of cDNA was used to establish standard curves for each gene to assess PCR efficiency and quantitative differences between samples. The qPCR amplification was performed using a MX 3005 real-time PCR system (Agilent) with Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent) as described previously (Kwiatkowska et al., 2013 (link)). The relative expression and fold-change of each target gene in mated relative to unmated was calculated according to the 2−ΔΔCT method incorporating PCR efficiency (Schmittgen and Livak, 2008 (link)) after normalization with the housekeeping genes rsp7, encoding ribosomal protein S7 (AGAP010592) and elongation factor gene (AGAP005128).
3
Optimized mRNA and EV RNA Reverse Transcription
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Messenger RNA (mRNA) isolated from tumor tissue and EV RNA isolated from plasma samples, was reverse transcribed into cDNA using the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific). The protocol was optimized to maximize cDNA yield. In the first part, 1.0 μL 50 μmol/L Oligo d(T)20, 1.0 μL 50 μmol/L random hexamers, 1.5 μL 10 mmol/L dNTP mix, and 1.0 μL 7-deaza-dGTP (New England BioLabs) were combined with template RNA in a 0.2 mL PCR reaction tube. Nuclease-free water was then added to reach a final reaction volume of 13 μL. The RNA–primer mix was then incubated at 65°C for 5 minutes, immediately followed by incubation on ice for 1 minute. In the second part, reverse transcription (RT) reaction mix was prepared by combining 4.0 μL 5× SSIV Buffer, 1.0 μL 100 mmol/L DTT, 1.0 μL ribonuclease inhibitor, and 1.0 μL SuperScript IV Reverse Transcriptase (200 U/μL) in a reaction tube to a final volume of 7 μL. The contents were briefly vortexed and centrifuged. The RT reaction mix was then added to annealed RNA from the first part of the protocol. Combined annealed RNA-RT reaction mix was then incubated at 23°C for 10 minutes, 53°C for 10 minutes, 80°C for 10 minutes. The resulting 20 μL cDNA was then purified using ethanol precipitation.
4
Quantitative RT-PCR Validation of Insecticide Resistance Genes
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A subset of eleven differentially transcribed genes was selected for quantitative real-time reverse transcription PCR validation (qRT-PCR). One microgram of RNA from three replicates of malathion resistant or permethrin resistant, non-exposed and Dongola strain mosquitoes were used to synthesize cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA) with oligo-dT20 (New England Biolabs, USA), according to the manufacturer's instructions. Primer sequences and efficiencies are detailed in Table S9. Standard curves of Ct values for each gene were generated using a five-fold serial dilution of cDNA to assess PCR efficiency. Reactions were performed using either a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems, USA) with PowerUp SYBR Green Master Mix (Applied Biosystems, USA) or a Stratagene Mx3005P Real-Time PCR system (Agilent Technologies) with LightCycler ® 480 SYBR Green I Master Mix (Roche, UK). cDNA from each sample was used as a template in a three-step reaction: . The relative expression level and Fold Change (FC) of each target gene from resistant field samples, relative to the susceptible laboratory strain (Dongola), were calculated using the 2 -ΔΔCT method (72), incorporating PCR efficiency. Two housekeeping genes, ribosomal protein S7 (RpS7: AARA000046) and ubiquitin (AARA016296), were used for normalisation.
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