The abundance of Asaia was assessed in duplicate using quantitative polymerase chain reaction (qPCR). The AsaH1_F and Asar_R primers were used to quantify the symbiont in each DNA template (Table S1 ). qPCR of Ribosomal Protein S7 (single copy gene) of both species (VectorBase ID: AFUN007153 in An. funestus; orthologous to An. gambiae: AGAP010592) was performed in parallel to normalize the Asaia load (Table S1 ). The reactions were run in a total volume of 10 μL, containing 5 μL of 2X Brilliant II SYBR® Green QPCR Master Mix (Agilent), 1 µM of each primer, 1 μL of nuclease-free water, and 2 μL template DNA. The reactions were run on an MX3005 real-time PCR system (Agilent) following a dissociation curve (95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s [40 (link)]. The standard curve of each gene was generated by diluting one-tenth of the DNA extracted from Asaia cultures and the purified PCR amplicons of the ribosomal protein S7 gene. Ct values of >35 were considered uninfected or undetectable. The normalized amount of bacterial DNA was estimated by the relative ratio of Asaia gene copy numbers to RSP-7 gene copy numbers. Mann Whitney and Kruskal Wallis non-parametric tests were used to compare the load of Asaia spp. between phenotype (alive and dead) and genotype, respectively, via GraphPad Prism 8.2.0.
Mx 3005 real time pcr system
Manufactured by Agilent Technologies
Sourced in United States
The MX 3005 is a real-time PCR system designed for quantitative analysis of nucleic acid samples. It features a flexible optical system and precise temperature control to enable accurate and reliable detection of gene expression and other targeted genomic sequences.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Oligo dt20, by New England Biolabs (2 mentions)
Brilliant 3 ultra fast sybr green qpcr master mix, by Agilent Technologies (2 mentions)
Rnase h, by New England Biolabs (2 mentions)
Prism 8, by GraphPad (1 mentions)
Brilliant 2 sybr green qpcr master mix, by Agilent Technologies (1 mentions)
4 protocols using mx 3005 real time pcr system
1
Quantitative Assessment of Asaia Symbiont
2
Validation of Detoxification Gene Expression
3
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).
4
Quantifying Cartilage Gene Expression
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Total RNA was isolated from cartilage tissues or chondrocytes by using TRIzol reagent. Isolated RNA was reverse transcribed with the use of a kit (Promega), and qPCR was performed with the Mx3005 real-time PCR system (Agilent). The expression of mRNAs relative to GAPDH was determined using the 2-ΔΔCT method44 (link). The following primers were used in this study:
COL2 forward: 5′-TGGACGATCACGAAACC-3′, reverse: 5′-GCTGCGGATGCTCTCAATCT-3′; aggrecan forward: 5′-ACTCTGGGTTTTCGTGACTCT-3′, reverse: 5′-ACACTCAGCGAGTTGTCATGG-3′; MMP13 forward: 5′-ACTGAGAGGCTCCGAGAAATG-3′, reverse: 5′-GAACCCCGCATCTTGGCTT-3′; ADAMTS5 forward: 5′-GAACATCGACCAACTCTACTCCG-3′, reverse: 5′-CAATGCCCACCGAACCATCT-3′.
COL2 forward: 5′-TGGACGATCACGAAACC-3′, reverse: 5′-GCTGCGGATGCTCTCAATCT-3′; aggrecan forward: 5′-ACTCTGGGTTTTCGTGACTCT-3′, reverse: 5′-ACACTCAGCGAGTTGTCATGG-3′; MMP13 forward: 5′-ACTGAGAGGCTCCGAGAAATG-3′, reverse: 5′-GAACCCCGCATCTTGGCTT-3′; ADAMTS5 forward: 5′-GAACATCGACCAACTCTACTCCG-3′, reverse: 5′-CAATGCCCACCGAACCATCT-3′.
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