RT-qPCR was executed as previously described (Yaniv et al., 2021 (link)). Reaction final volume is 20 µL with primers concentration of 0.5 µM and probe concentration of 0.2 µM. The reaction contained 5 µL of RNA sample and ROX as a reference dye. Reaction steps were executed according to manufacture recommended protocol (One Step PrimeScript III RT-qPCR mix RR600 TAKARA, Japan) using Applied Biosystems Thermocycler (Thermo Scientific). For reverse transcription reaction, the reaction holding stage carried out for 7 min at 52 ℃ followed by 10 s at 95 ℃. For PCR reaction, 45 cycles were performed at 95 ℃ for 5 s followed by 30 s at 60 ℃. Each RT-qPCR run included relevant quality controls, Non template control (NTC) and MS2 phage detection for wastewater RNA sample (Dreier et al., 2005 (link)). MS2 addition was meant for inhibitors assessment through calculated recovery percentage (appear in Table S4, values were calculated using Fig. S1). Ct value initially converted to RNA copies per μL (in the RNA sample) based on the standard curves (as described in the next section). Duplicate were averaged for each target gene separately. The gene copies per L of wastewater sample were calculated according to Eqs. (1) and 2 for Raw or Concentrated sample respectively.
Applied biosystems thermocycler
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The Applied Biosystems thermocycler is a laboratory instrument used for the amplification of DNA or RNA samples through the polymerase chain reaction (PCR) process. It precisely controls the temperature, duration, and cycling of the samples to facilitate the replication of genetic material.
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5 protocols using applied biosystems thermocycler
1
RT-qPCR for Wastewater SARS-CoV-2 Monitoring
2
MLVA Genotyping of M. hyopneumoniae from PCR-Positive Samples
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For the samples positive for M. hyopneumoniae by real-time PCR, the number of copies was elaborated based on a standard reference curve. These samples were genotyped after with MLVA. Briefly, the method consisted of four conventional PCR for the amplification of Locus 1, Locus 2, P97-RR1, and P97-RR2 genes in accordance with the scheme proposed by Charlebois et al. [13 (link)] and Tonni et al. [17 (link)]. The PCR were performed with an Applied Biosystems thermocycler (Thermo Fisher Scientific), and the PCR products were examined by capillary electrophoresis (QIAxcel; Qiagen, Hilden, Germany). In case of unclear results, further evaluation was performed with a 2.0% high-resolution agarose gel run at 100 V for 2 h and visualized under ultraviolet light. For each VNTR locus, the estimated number of tandem repeats was calculated according to the allele calling table (Additional file 1 ). Each VNTR type was defined based on the number of repeats per locus. The VNTR type nomenclature was assigned following the chronological order of identification at the IZSLER laboratory.
The presence of one or more M. hyopneumoniae VNTR types per sample was defined as a single (SN) or mixed (MX) infection, respectively.
The presence of one or more M. hyopneumoniae VNTR types per sample was defined as a single (SN) or mixed (MX) infection, respectively.
3
Wastewater SARS-CoV-2 RT-qPCR Quantification
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The Wastewater's RNA were subjected to an RT-qPCRs assays as previously described (Yaniv et al., 2021a (link), Yaniv et al., 2021b (link)). Briefly, reaction final volume is 20 μL with primers and probes concentration of 0.5 μM and 0.2 μM respectively. Each reaction mixture was added with 5 μL of RNA sample. Assay steps were preformed according to manufacture recommended protocol (One Step PrimeScript III RT-qPCR mix RR600 TAKARA, Japan) using Applied Biosystems Thermocycler (Thermo Scientific). RNA copy number per Liter of wastewater for raw sample was calculated using an equation available in the SI file (page 17). Each RT-qPCR assay run included relevant quality controls (such as: Non template control (NTC) and MS2 phage detection for wastewater RNA sample (Dreier et al., 2005 (link))).
4
Pancreatic RNA Extraction and cDNA Synthesis
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Approximately 40–50 mg of pancreatic tissue was used for total RNA extraction using RNAiso Plus (Takara). Quantity and quality of total RNA were determined in the ratio of OD260 nm/OD280 nm, which was expected to be between 1.80 and 2.0, using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The integrity of RNA was confirmed by staining 18s and 28s rRNA bands on 1% agarose gel. Total RNA (1 μg) was treated with gDNA Eraser at 42°C for 2 min (Takara) to remove genomic DNA contamination, according to the manufacturer's protocol. Reverse transcription was performed using the Prime Script RT Reagent Kit (Takara), according to the manufacturer's protocol, in an Applied Biosystems thermocycler (Thermo Fisher Scientific) at 37°C for 15 min and at 85°C for 5 min and then cooled to 4°C. cDNA samples were stored at −20°C for Reverse transcription‐qPCR analysis.
5
Quantitative PCR Analysis of Gene Expression
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Total RNA was extracted using TRIzol (Life Technologies; Thermo Fisher Scientific, Inc.) and subjected to RT with a Reverse Transcriptase kit (Fermentas; Thermo Fisher Scientific, Inc.). The sequences of the human specific primers used for PCR were as follows: ABCG2, forward 5′-TCAATCAAAGTGCTTCTTTTTTATG-3′ and reverse 5′-TTGTGGAAGAATCACGTGGC-3′; Nrf2, forward 5′-ACACGGTCCACAGCTCATC-3′ and reverse 5′-TGCCTCCAAAGTATGTCAATCA-3′; and glyceraldehyde 3-phosphate dehydrogenase, forward 5′-ATGTCGTGGAGTCTACTGGC-3′ and reverse 5′-TGACCTTGCCCACAGCCTTG-3′ (IDT Shanghai Co. Ltd., Shanghai, China). The PCR parameters were as follows: Initial denaturation at 95°C for 2 min, followed by 35 cycles of annealing at 58°C for 45 sec, extension at 72°C for 2 min and a final extension at 72°C for 7–10 min. The reaction was performed in an Applied Biosystems thermocycler (Thermo Fisher Scientific, Inc.). The amplified products were analyzed on 1.5% agarose gel electrophoresis and visualized with ethidium bromide, using a gel image documentation device (Bio Basic Canada, Inc., Markham, ON, Canada). The intensity of the DNA bands from three independent experiments was measured with ImageJ version 3.2 (https://imagej.nih.gov/ij/ ).
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