Ears were harvested 15 days post-infection after euthanizing the mice. Whole ears were cut and washed in ethanol for 5 minutes followed by digestion in trizol (ZYMO Research). DNA was isolated from ears using the DNeasy Blood and Tissue Kit (Qiagen, 69506). Parasite DNA was quantified using previously described primers that amplify the kinetoplastid DNA (kDNA): forward– 5’- CCTATTTTACACCAACCCCCAGT-3’ (JW11); reverse—5’-GGGTAGGGGCGTTC TGCGAAA-3’ (JW12) [39 ]. The murine housekeeping genes used were GAPDH amplified using previously described primers: GAPDH-F 5’- TTTGATGTTAGTGGGGTCTCG-3’; GAPDH-R 5’-AGCTTGTCATCAACGGGAAG-3’ [40 (link),41 (link)]. PCR was performed using the Applied Biosystems ViiA Real Time PCR System in a 20 μl reaction mix with 10 μl SYBR Green master mix (Applied Biosystems), 1 μmol each of the forward and reverse primers and 50ng of the template. The PCR was run for 40 cycles at 95°C for 10 sec, 60°C for 15 sec and 72°C for 30 sec. Parasite Ct were normalized against mouse GAPDH Ct and the numbers of parasites were quantified using the standard curve of mouse ear tissues with known numbers of Leishmania parasites (limit of linear detection ranges from 102 to 106 parasites per ear).
Viia real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The ViiA Real-Time PCR System is a high-performance real-time PCR instrument designed for quantitative gene expression analysis, genotyping, copy number variation, and other applications requiring sensitive and precise real-time PCR detection. The system features a 384-well block format, advanced optics, and intuitive software for efficient data analysis and reporting.
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Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Power sybr green rna to ct 1 step kit, by Qiagen (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Tri reagent, by Merck Group (3 mentions)
Tissuelyser 2, by Qiagen (3 mentions)
Qiaamp dna blood mini kit, by Qiagen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Tri reagent, by Qiagen (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Trizol, by Zymo Research (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Taqman genotyping assay, by Thermo Fisher Scientific (1 mentions)
Copycaller software, by Thermo Fisher Scientific (1 mentions)
384 well plate, by Thermo Fisher Scientific (1 mentions)
Epmotion m5073, by Eppendorf (1 mentions)
26 protocols using viia real time pcr system
1
Quantifying Leishmania Parasite Burden
2
Quantifying CFHR1/CFHR3 Copy Number
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DNA was extracted from whole blood samples using QIAamp DNA Blood Mini Kits (Qiagen, Hilden, Germany). Quantitative real-time polymerase chain reaction (PCR) was performed using the ViiA Real-Time PCR System (Applied Biosystems, Foster City, CA). Copy number variation within the CFHR3 and CFHR1 genes was assessed using the Taqman Copy Number Real-Time Detection System (Applied Biosystems). Copy number variation calls were determined using the Copy Caller Software (Applied Biosystems). Assay readings were normalized to control samples, and the values were presented as mean ± SD. All probes were validated using genomic DNA from healthy controls with either heterozygous or homozygous polymorphic deletion of the CFHR1 and CFHR3 genes. The CFHR1 gene copy number in the renal transplant cohort was inferred from the rs6677604 genotype, which is in linkage disequilibrium with the CFHR1 and CFHR3 gene copy number.16 (link) Genotyping was performed using the Taqman genotyping assays (Applied Biosystems).
3
Quantitative Real-Time PCR Analysis
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Trizol (Invitrogen) was used to extract mRNA from pellets as per the manufacturer’s protocol. The RNA concentration was measured using spectrophotometer (Nanodrop 1000, Thermo Scientific). Complementary DNA (cDNA) was synthesized from the mRNA template using SuperScript III RT and oligo(dT)20 kit (Invitrogen) as per the manufacturer’s instructions. The quantitative polymerase chain reaction (qPCR) was performed using Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). The primers (5’ to 3’, Geneworks) listed in S1 Table were used.
The qPCR reactions mixes were aliquoted using a liquid handler (epMotion M5073, Eppendorf). SYBR Green master mix was dispensed into 384-well plates (Applied Biosystems) and combined with cDNA template. The qPCR reaction was performed using ViiA real time PCR system (Applied Biosystems). The qPCR reaction was initiated with a 2-minute 50°C hold, followed by a 3-minute 95°C hold, and then proceeded with 40 cycles of 15-second at 95°C, 30-second at 60°C. The qPCR results were analyzed using ΔCt method and the gene expression was normalized to the geometric mean of two housekeeping genes (cyclophilin A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)).
The qPCR reactions mixes were aliquoted using a liquid handler (epMotion M5073, Eppendorf). SYBR Green master mix was dispensed into 384-well plates (Applied Biosystems) and combined with cDNA template. The qPCR reaction was performed using ViiA real time PCR system (Applied Biosystems). The qPCR reaction was initiated with a 2-minute 50°C hold, followed by a 3-minute 95°C hold, and then proceeded with 40 cycles of 15-second at 95°C, 30-second at 60°C. The qPCR results were analyzed using ΔCt method and the gene expression was normalized to the geometric mean of two housekeeping genes (cyclophilin A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)).
4
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from cell pellets using an RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) with a genomic DNA (gDNA) Eliminator spin column. To remove contaminating genomic DNA, 1 µg RNA was DNase treated prior to cDNA synthesis with Superscript III (Invitrogen). qRT-PCR was performed using the AB7500 FAST sequence detection thermal cycler (Applied Biosystems, Foster City, CA), or the ViiA Real-Time PCR system (Applied Biosystems) with SYBR Green PCR Master Mix (QIAGEN) using primers listed in Table S11 . A negative control (water instead of template) was used in each real-time plate for each primer set. All real-time experiments were performed in triplicate. Baseline and threshold values (Ct) were obtained using ABI 7500 Prism and the relative expression of mRNA was calculated using the comparative 2−ΔΔCt method (Livak & Schmittgen, 2001 (link)). Expression was normalized to the housekeeping gene ribosomal protein L32 (RPL32). Statistical analyses were performed using GraphPad Prism v.6.01 software (GraphPad Software, Inc., San Diego, CA). Student’s t-test or Mann–Whitney-Wilcoxon tests were used to assess the statistical significance of all the direct comparisons.
5
Quantitative Real-Time PCR for Gene Expression
6
RT-qPCR for Viral M Gene
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Total RNA was isolated from viral stocks using the QIAamp viral RNA minikit (Qiagen) and reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) with random hexamers. Real-time quantitative PCR (RT-qPCR) for the viral M gene was performed with TaqMan probe 5′-FAM-TYACGCTCACCGTGCCCAGTG-MGBNFQ-3′ (FAM, 6-carboxyfluorescein) (Life Technologies). The primer sequences were AAGACAAGACCAATYCTGTCACCTCT (FWD) and TCTACGYTGCAGTCCYCGCT (REV). Data were analyzed on the Applied Biosystems ViiA real-time PCR system.
7
RNA Isolation and Quantitative PCR
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For RNA isolation, bacteria were grown in 20 ml of THY, harvested in the exponential growth phase (optical density at 600 nm = 0.3) and quickly frozen in liquid nitrogen. As described by Pappesch et al. [22 (link)], RNA was isolated with the Direct-zol RNA MiniPrep Kit (Zymo Research), subsequent acid phenol:chloroform:isoamyl alcohol (125:24:1) extraction and TURBO DNase treatment. cDNA was generated with the SuperScript first-strand synthesis system for RT-PCR (Thermo Fisher Scientific). SYBR green (Thermo Fisher Scientific)-based quantitative real time PCR was carried out on a ViiA Real-Time PCR System (Applied Biosystems). The 5S rRNA gene was used as a housekeeping gene. Primers used for S5nA and 5S cDNA detection are listed in Table 1 .
8
RT-qPCR for Target Gene Expression
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To validate the transcriptomic results and detect target gene expression precisely, RT-qPCR was applied to quantify mRNA levels. Briefly, 500 ng of RNA was subjected to reverse transcription using PrimeScript RT Master Mix (TaKaRa Biomedicals, Kusatsu, Japan), and subsequently, the synthesized cDNA was used for RT-qPCR with TB Green Premix Ex Taq II (TaKaRa) and paired primers on a ViiA real-time PCR system (Applied Biosystems, CA, USA). The reaction of each sample was performed in duplicate.
The relative expression of target genes was standardized to the GAPDH mRNA levels, and fold changes between groups were calculated based on the 2-△△Ct method. All paired primer sequences are listed in Table S1.
The relative expression of target genes was standardized to the GAPDH mRNA levels, and fold changes between groups were calculated based on the 2-△△Ct method. All paired primer sequences are listed in Table S1.
9
Quantitative gene expression analysis
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Analysis of gene expression using qPCR was performed as previously described [49 (link)]. The reactions were performed on ViiA Real-Time PCR System (Applied Biosystems) with Taqman Fast Universal PCR Master Mix (Life Technologies). The primer sets for all the genes was purchased from Life Technologies. The expression level of the genes was calculated using standard ΔΔCt method, with GAPDH as internal control. All reactions were performed with at least 3 replicates.
10
TAK-243 Treatment Time-Course in MiaPaCa-2 Cells
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MiaPaCa-2 cells were grown in 6-well dishes and TAK-243 was added at a final concentration at 300 nM for 0, 6, 8 and 10 h. Total RNA was extracted with Qiagen RNeasy kit and cDNA was synthesized using High-Capacity Reverse Transcription kit (Applied Biosystems). Quantitative PCR was performed using SYBR Green Master Mix (Bio-Rad) on a ViiA real-time PCR system (Applied Biosystems) with denaturation at 95 °C for 15 s; 55 °C for 30 s; 72 °C for 30 s. mRNA expression level was evaluated using the ΔΔ Ct method.
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