cDNA expression was amplified with QuantStudio 5 qPCR machine (Thermo Fisher, MA, USA) using LightCycler 480 SYBR Green I Master (Roche, Mannheim, Germany) and specific forward miRNA primers and universal reverse miRNA primers (Genedragon, Taipei, Taiwan). All samples were assayed in triplicate. Each miRNA expression was normalized to cel- miR-39 and calculated as 2−ΔCt, ΔCt = (Ct[miR]−Ct[cel-miR-39]) [26 (link)].
Quantstudio 5 qpcr machine
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 5 qPCR machine is a real-time PCR system designed for gene expression analysis and quantification. It features a 96-well block format and supports a wide range of sample volumes. The system is capable of performing relative and absolute quantification, as well as high-resolution melt analysis.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Rlt lysis buffer, by Qiagen (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Sybr green fluorescent dye, by Thermo Fisher Scientific (2 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini column, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Perfecta sybr green fastmix and low rox, by Quantabio (1 mentions)
Qscript cdna synthesis kit, by Quantabio (1 mentions)
Fast sybr green, by Thermo Fisher Scientific (1 mentions)
25 protocols using quantstudio 5 qpcr machine
1
Quantitative miRNA expression analysis
2
Rapid SARS-CoV-2 Detection from Saliva
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SARS-CoV-2 RNA was isolated from crude saliva samples using QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany; #52906), following manufacturer’s recommendations. RT-PCR was performed based on CDC’s protocol, which targets SARS-CoV-2 gene N (Centers for Disease Control and Prevention, 2020). RT-PCR reactions (12 μL total volume) contained TaqMan® Fast Virus 1-Step Master Mix (Thermo Fisher, Waltham, MA, USA; #4444432) and a primer/probe mix (500 nM forward primer, 500 nM reverse primer, 125 nM probe; 2019-nCoV_N1, IDT, Coralville, IA, USA; #10006600), and were cycled in a QuantStudio 5 qPCR machine as per manufacturer’s recommendations (Thermo Fisher, Waltham, MA, USA). Absolute quantification of viral RNA copies was performed via standard curve assays with 2019-nCoV_N_Positive Control (IDT, Coralville, IA, USA; #10006625), in triplicates.
3
Isolation and Analysis of RNA from ESCs and Embryos
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RNA was isolated directly from ESCs by scraping in RLT lysis buffer (Qiagen) containing 1:100 β-mercaptoethanol (Sigma), or RLT was added to equal numbers of ESCs for CNN approaches. RNA was purified and DNase I-treated according to the manufacturer's instructions using RNeasy mini kits (Qiagen). For embryo inhibitor experiments, two-cell embryos were flushed at 46 h after hCG and cultured in KSOMaa medium containing either 1 µM CX-5461, 1 µM BMH-21, or 0.1% DMSO in a four-well dish. Culture in inhibitors began after 1 h for a period of 8 or 24 h. Equal numbers of embryos per experimental condition were lysed in 75 µL of buffer RLT prepared as above, and the RNA was isolated according to RNeasy micro kits (Qiagen). In ESCs and embryos, cDNA synthesis was performed with up to 1 µg of DNase-treated RNA using a high-capacity RNA-to-cDNA kit (Thermo Fisher Scientific), and qRT-PCR was performed with SYBR Green (KAPA) on a QuantStudio 5 qPCR machine (Thermo Fisher Scientific). qRT-PCR data were normalized to two housekeeping genes (Rpl7/H2A), unless a cell number normalization (CNN) approach was used as detailed in the legend for Figure 4 , A, G, and H. Primer sequences are described in Supplemental Table S2 .
4
Thermal Shift Assay for LsrK Screening
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Compounds were added to qPCR plates by Echo acoustic dispenser with a DMSO backfill to maintain a constant final concentration of DMSO in each well (the assay is tolerant up to 8% DMSO with no effect on Tm of LsrK). LsrK protein was added at a concentration of 1 µg per well in a volume of 10 µL with 1× Thermo thermal shift dye or 5× Sypro Orange dye (used for aurin tricarboxylic acid as this compound fluoresces with Thermo dye in 25 mM TEA, 200 µM MgCl2, pH 7.1). Appropriate DMSO-only negative controls and 1 mM ATP/DMSO positive controls were included in each test occasion. Using an Applied Biosystems Quant Studio 5 qPCR machine (Thermo Fisher Scientific, Waltham, MA, USA) the following thermal shift protocol was used: step 1—hold at 25 °C for 1 min; step 2—heat at 0.05 °C/s dissociation then hold at 99 °C for 1 min. Filter setting for Thermo dye: Ex/Em 580/623 nm and for Sypro Orange dye: Ex/Em 470/520 nm.
5
Quantifying Gene Expression via qPCR
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RNA extraction, reverse transcription, and quantitative PCR (qPCR) was generally performed as described previously.103 (link) Cultured cells were directly lysed in the culture plate, or alternatively, were purified via FACS and then lysed, using 350 μL of RLT Plus Buffer. RNA was then extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s protocol. 300 ng of total RNA was reverse transcribed into cDNA for qPCR using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. qPCR was performed in 384-well format as previously described,103 (link) using gene-specific forward and reverse primers, as well as the SensiFAST SYBR Green Lo-ROX Kit (Thomas Scientific), on a QuantStudio 5 qPCR machine (Thermo Fisher). qPCR primer sequences are provided in Table S4 . Expression of all genes was normalized to the levels of the reference gene YWHAZ.
6
Quantitative Analysis of DNA Repair Genes
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Total RNA was obtained from R182 and TARA cell pellets using the Total RNA Kit from IBI Scientific (Dubuque, IA, USA). RNA yield and quality was assayed using absorbance spectra on a NanoDrop 2000 (Thermo Fisher). cDNA was reverse transcribed using the qScript cDNA synthesis kit from Quantabio (Beverly, MA, USA). Quantitative PCR was run on an Applied Biosystems Quantstudio 5 qPCR machine (Thermo Fisher), using PerfeCTa SYBR Green FastMix and Low ROX (Quantabio) in 10 μL reactions. PCR was run for 40 cycles, followed by melt curve analysis. Beta actin was used as a housekeeping gene. Relative gene expression levels were calculated using the 2−ΔΔCt method. Primers were obtained from Integrated DNA Technologies (Coralville, IA, USA), and sequences were as follows: BRCA2-Fd, 5′-AAAACGTTGAGCTGTTGCCA-3′; BRCA2-Rv, 5′-TGTGTTTTGGTTGAATTGTACCTT-3′; RAD51 Fd, 5′-CCAGACCCAGCTCCTTTACC-3′; RAD51 Rv, 5′-CACTGCGACACCAAACTCATC-3′; BRCA1 Fd, 5′-GGCTATCCTCTCAGAGTGACATTT-3′; BRCA1 Rv, 5′-GCTTTATCAGGTTATGTTGCATGGT-3′; Actin Fd, 5′-TTCCTGGGCATGGAGTCC-3′; and Actin Rv, 5′-CAGGTCTTTGCGGATGTCC-3′.
7
Isolation and Profiling of Pancreatic Macrophages
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Isolated fractions of exocrine and endocrine macrophages pooled from two pancreata per observation were subjected to mRNA isolation using a commercial kit (Single Cell RNA Purification Kit, Norgen Biotek). Synthesis to cDNA was done using the High Capacity cDNA Synthesis kit (ThermoFisher). Custom oligos were from ThermoFisher (primer sequences in Supplemental Table 2
8
RNA Isolation and Quantitative PCR
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RNA was isolated using the RNeasy Mini Kit (Qiagen, Cat. #74104) as per the manufacturer’s protocol. RNA was quantified and quality-checked using TECAN plate reader. RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat. #4368813) as per the manufacturer’s protocol. Quantitative PCR was performed using the PrimeTime Gene Expression Master Mix (IDT, Cat. #1055771) for probe-based RT-qPCR. See supplementary table 1 for probe primer sequences. A 10 ng cDNA was used for each reaction and Gapdh was used as an internal endogenous control. Fold changes were calculated using the ΔΔCT method relative to the expression levels in MEFs. All qPCR reactions were performed using the QuantStudio5 qPCR machine (Thermo Fisher Scientific, Cat. #A34322).
9
Analyzing Protein Thermal Stability
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Melting temperatures (Tm) of PtUGT1 WT and variants were measured by DSF using the Protein Thermal Shift Dye Kit (ThermoFisher Scientific, Germany) and a QuantStudio5 qPCR machine (ThermoFisher Scientific, Germany). Dye solution (1000x) was diluted to final (2x) in Buffer 2x (100 mM HEPES pH7, 100 mM NaCl). 10 μL of dye solution 2x was mixed with 10 μL of 0.8 mg/mL protein sample and pipetted in the qPCR multi-well plate. The plate was centrifuged 30 s at 142 x g and transferred to the qPCR machine. The protocol initiates with 2 min incubation at 25 °C, followed by a temperature increase of 0.05 °C/s up to 99 °C, and a final incubation of 2 min at 99 °C. Measurements were carried out in triplicate. Raw data was obtained with QuantStudio Design & Analysis Software 1.5.1 and further analyzed with Protein Thermal Shift™ Software v1.4 (Fig. 2C ).
10
Quantitative Real-Time PCR Analysis
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Three hundred naograms of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. qPCR was performed in 384-well format31 (link) on a QuantStudio 5 qPCR machine (Thermo Fisher). Expression of all genes was first normalized to the levels of the reference gene YWHAZ, and then plotted relative to the levels of YWHAZ (i.e., 1.0 = equivalent to YWHAZ). Forward and reverse primer sequences used to detect the expression of the respective genes are provided in Supplementary Table 1 .
All qPCR experiments were performed once. For each qPCR experiment, two biological replicates (two different wells in the same culture plate) were analyzed; qPCR was then performed on two technical replicates for each biological replicate (i.e., 2 biological replicates × 2 technical replicates = 4 replicates altogether). Plotted qPCR data represents the average of the 4 replicates, and standard error is shown.
All qPCR experiments were performed once. For each qPCR experiment, two biological replicates (two different wells in the same culture plate) were analyzed; qPCR was then performed on two technical replicates for each biological replicate (i.e., 2 biological replicates × 2 technical replicates = 4 replicates altogether). Plotted qPCR data represents the average of the 4 replicates, and standard error is shown.
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