Dust samples from the farms and the cave environment that tested positive in real-time PCR with Cq below 31 were genotyped by a 10-loci single nucleotide polymorphism (SNP) discrimination test using real-time PCR as described elsewhere [22 (link)]. Briefly, 10 real-time PCR reactions were performed per dust DNA sample, each including two primers and two MGB TaqMan probes (labelled with VIC and FAM at the 5’ end, respectively) (Life Technologies S.A., Alcobendas, Spain) to detect point mutations at each of the 10 sites. Coxiella burnetii Nine Mile strain was used as a positive control.
Mgb taqman probes
Manufactured by Thermo Fisher Scientific
Sourced in Spain
MGB TaqMan probes are oligonucleotide-based fluorescent detection reagents designed for use in real-time PCR assays. They incorporate a minor groove binder (MGB) and a fluorescent reporter dye, enabling efficient and specific detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis supermix kit, by Thermo Fisher Scientific (2 mentions)
Rt pcr rotor gene q, by Qiagen (2 mentions)
E z n a forensic dna kit, by Omega Bio-Tek (2 mentions)
Superscript 2 rt enzyme, by Roche (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Roche (1 mentions)
Abi7300 platform, by Thermo Fisher Scientific (1 mentions)
Taqman genotyping master mix, by Thermo Fisher Scientific (1 mentions)
Uv 1700 pharmaspec, by Shimadzu (1 mentions)
Steponeplus real time pcr system, by Agilent Technologies (1 mentions)
Brilliant 2 qpcr master mix, by Agilent Technologies (1 mentions)
Abi prism 7900 sequence detector, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
9 protocols using mgb taqman probes
1
Genotyping Coxiella burnetii via SNP Assay
2
Quantitative Real-Time PCR for Gene Expression
3
Caffeine Synthase Genotyping Protocol
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Genomic DNA was extracted from collected leaves using a CTAB based protocol (Doyle and Doyle 1990) . After extraction, DNA was quantified by absorbance under UV light at 260 and 280 nm on a spectrophotometer PharmaSpec UV-1700 (UV-Visible Spectrophotometer SHIMADZU), according to Sambrook et al. (1989) . A conventional PCR was performed to verify overall DNA quality using control actin specific primers as previously described (Maluf et al. 2009) .
Genotyping was performed by quantitative PCR using a combination of primers and specific MGB TaqMan probes (Life Technologies). Caffeine synthase specific primers and fluorescent probes annealing to a region with two SNPs were used for allelic discrimination using an ABI7300 platform (Applied Biosystems). Sequences and fluorescence used are listed in Online resource 2. For each reaction 100 ng of DNA were used, and amplification conditions were as suggested on the commercial TaqMan® Genotyping Master Mix (Thermo Scientific) and equipment manuals. All reactions were performed in duplicate, and were repeated two times for each genotype. Post-running analyses were performed using the software 7300 System SDS (Applied Biosystems).
Genotyping was performed by quantitative PCR using a combination of primers and specific MGB TaqMan probes (Life Technologies). Caffeine synthase specific primers and fluorescent probes annealing to a region with two SNPs were used for allelic discrimination using an ABI7300 platform (Applied Biosystems). Sequences and fluorescence used are listed in Online resource 2. For each reaction 100 ng of DNA were used, and amplification conditions were as suggested on the commercial TaqMan® Genotyping Master Mix (Thermo Scientific) and equipment manuals. All reactions were performed in duplicate, and were repeated two times for each genotype. Post-running analyses were performed using the software 7300 System SDS (Applied Biosystems).
4
Gene Expression Quantification
5
Quantitative Real-Time PCR Analysis
6
Quantitative Real-Time PCR Analysis
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Mouse kidneys and livers were lysed in TRIzol, and the total RNA was purified by chloroform extraction and isopropanol precipitation. RT was performed from 500 ng of total RNA with the Takara PrimeScript RT Reagent Kit (Ozyme, Saint Quentin en Yvelines, France). Primers and MGB-Taqman probes were purchased from ThermoFisher Scientific (Table S2 ). The PCR reaction mixture was prepared using the Brilliant II QPCR Master Mix (Agilent Technologies, Les Ulis, France). All PCR reactions were performed in a StepOnePlus Real-Time PCR System (Agilent Technologies, Thermo Fisher Scientific). The data were acquired and analyzed with the StepOne software (Agilent Technologies). Target gene expression was normalized on the basis of the GUSB content of each sample, and was subsequently normalized to a basal mRNA level with the equation N target = 2ΔCt sample, where ΔCt is the Ct value of the target gene minus the Ct value of the HPRT gene. The results are reported as “normalized mRNA levels”—i.e., the N target value divided by the N target value of the smallest quantifiable amount of target gene mRNA (target gene Ct value = 35).
7
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from approximately 25 mg of cortical tissue using Tri Reagent (Sigma-Aldrich) and subsequently quantified spectrophotometrically at 260 nm with RNA purity being determined as the ratio of 260/280 nm readings. Thereafter, first-strand cDNA synthesis was carried out according to standard methodology. Transcripts were quantified by TaqMan PCR using an ABI prism 7900 sequence detector (Applied Biosystems Inc., Foster City, CA, USA) with 2 μl of cDNA, 18 μM of each primer, 5 μM probe and Universal TaqMan 2x PCR Mastermix (Roche). Each sample was run in duplicate. Primers and MGB TaqMan probes (Thermo Fisher Scientific) were chosen where possible such that probes span over exon–exon boundaries to avoid genomic amplification (Supplementary Table S2 ). Hydroxymethylbilane synthase was used as internal control, and all genes of interest were labelled with the fluorescent reporter 5-carboxyfluorescein. Thermal cycling conditions: 10 min at 95 °C, followed by 40 cycles at 95 °C for 10 s then 60 °C for 30 s. The relative gene expression in the Psmc1fl/fl;CaMKIIα-Cre group compared with the control was calculated using the 2−ΔΔCt method.
8
Genotyping of eNOS Polymorphisms
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Approximately 2 mL of saliva were collected in PBS containing tubes. Genomic DNA was isolated with E.N.Z.A. Forensic DNA Kit (Omega bio-tek) according to manufacturer's instructions. Genotyping of T786C (rs2070744) and G894T (rs1799983) eNOS polymorphisms were performed by real time polymerase chain reaction (RT-PCR) using specific TaqMan probes MGB® (Applied Biosystems). All PCR amplifications were carried out in a RT- PCR Rotor Gene Q (Qiagen). The amplification parameters were as follows: 10 min initial denaturation at 95 °C, 40 cycles for 15 s at 92 °C, 90 s at 60 °C, and 1 min final extension at 60 °C. For the 27-bp intron 4VNTR eNOS polymorphism genotyping, conventional PCR was performed with 200 ng of DNA followed by gel electrophoresis. There are three different alleles for this polymorphism: allele “a” of 453 bp, “b” of 480 bp, and “c” of 507 bp. The amplification parameters were as follows: 15 min initial denaturation at 96 °C, 35 cycles for 30 s at 95 °C, 20 s at 59 °C, and 45 s at 72 °C, and 10 min final extension at 72 °C. Primers used for PCR amplification were: forward 5′- TGGAAAGGTAGGGGGACTG-3′ and reverse 5′- GGTCACAGGCGTTCCAGTA-3′. PCR products were loaded in a 4% agarose gel and visualized under UV light.
9
Genotyping of OPRM1 and COMT Polymorphisms
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Approximately 2 ml of saliva was collected in tubes containing 6 ml of PBS. Once the saliva sample was taken, it was stored at − 80 °C until its processing. Genomic DNA was isolated using the E.N.Z.A. Forensic DNA kit (Omega bio-tek), according to the manufacturer’s instructions. Real-time polymerase chain reaction (RT-PCR) analysis was used to genotype OPRM1 (rs1799971, A118G) and COMT (rs4680, G472A) gene polymorphisms. All PCR amplifications were carried out in a RT-PCR Rotor Gene Q (Qiagen), using specific TaqMan probes MGB® (Applied Biosystems). The amplification parameters were as follows: initial 10 min denaturation at 95 °C, 45 cycles for 15 s at 92 °C, 90 s at 60 °C, and 1 min final extension at 60 °C.
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