Total RNA was isolated from tissues using QIAzol lysis reagent (79306; Qiagen) as described in the manufacturer’s protocol. cDNA was synthesized from 2 µg of total RNA using a cDNA synthesis kit (K1622; Thermofisher Scientific). qPCR was carried out using qPCR Master Mix (M3003L; New England Biolabs) in ViiA 7 Real-Time PCR system (Applied Biosystems). 36B4 or GAPDH was used for normalization. The primer sequences used in the study are listed in the Supplementary Table 3 .
Qpcr master mix
Manufactured by New England Biolabs
Sourced in United States
The QPCR Master Mix is a ready-to-use solution for quantitative polymerase chain reaction (qPCR) experiments. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, and buffer, to perform real-time PCR amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Amv rt kit, by New England Biolabs (1 mentions)
Rt pcr machine, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Rotor gene q real time pcr, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Tripure isolation reagent, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qpcrbio cdna synthesis kit, by PCR Biosystems (1 mentions)
Am2222, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Dnase 1, by Quanta Biosciences (1 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (1 mentions)
7 protocols using qpcr master mix
1
Comprehensive RNA Extraction and qPCR Analysis
2
Quantitative RT-PCR Analysis of Leishmania Transcripts
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Total RNA was isolated from Leishmania cells using TRIzol reagent (Life Technologies) and dissolved in nuclease-free molecular grade water in the presence of RNase A Inhibitor (NEB) and stored at −80 °C. Purity of RNA was checked by 260/280 ratio in a spectrophotometer. Followed by DNase I (Roche) treatment, 1 μg of total RNA was denatured and mixed with 1 mM dNTPs, 1× RT buffer and reverse transcriptase, gene-specific primers as recommended in AMV RT kit (NEB), and incubated at 42 °C for 30 min for cDNA synthesis. cDNA preparation confirmed by PCR. Next for quantitative PCR analysis, 100 ng of cDNA and gene-specific primers were mixed with 1× premixed solution Luna qPCR master mix (NEB) of Taq polymerase, dNTPs, and buffer in a 20 μl reaction volume. The RT-PCR analysis was done in a BioRad RT-PCR machine. SYBR green dye binds to double-stranded DNA. The Ct values were calculated as fold change in expression.
3
Quantitative Gene Expression Analysis
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The total RNA was isolated from cells using Tripure Isolation Reagent (Roche Diagnostics GmbH, Mannheim, Germany) following manufacture’s instruction. Reverse transcription was performed using iScript cDNA Synthesis Kit (#1708891, Bio-Rad, Hercules, CA, USA) for cDNA synthesis and the quantitation of gene expression was achieved from Luna® Universal quantitative Polymerase Chain Reaction (qPCR) Master Mix (#M3003, New England Biolabs, Ipswich, MA, USA) following manufacture protocol. Quantitative PCR reactions were performed using a QIAGEN Rotor Gene Q Real-Time PCR. The primer sequences (5′–3′; forward, reverse) used in qPCR was showed in Table A1 . Amplification was achieved for 40–50 cycle following enzyme activation step in 95 °C for 1 min hold, denaturation in 95 °C for 15 s, and extension step in 60 °C for 30 s. The expression of target genes was normalized to internal control β-actin. The fluorescence signal was detected at the end of each cycle. Melting curve analysis was used to confirm the specificity of the products. The data were calculated using the comparative methods of the −ΔCt (-delta Ct) or 2−ΔΔCt (standardized mRNA level).
4
Transcriptional Analysis of Yeast Stress Response
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The FY73 (wild-type) and znf1Δ strains were grown in YPD at 30°C overnight until an OD600 of 0.1 and regrown to an OD600 of approximately 0.6. Then, 10% (vol/vol) ethanol was added to cell culture, and sampling was performed at 25 min and 6 h. For growth under high-glucose conditions, the BY4742 (wild-type) and znf1Δ strains were grown in YPD at 30°C overnight. Next, cells were regrown to the mid-log phase and transferred to YP medium containing 20% glucose (wt/vol) for additional 24 h. Yeast cells were harvested and washed twice using distilled water. Then, the total RNA was extracted by the phenol-chloroform method (74 (link)) and purified using the RNeasy minikit (Qiagen, Hilden, Germany). cDNA synthesis was carried out with the qPCRBIO cDNA synthesis kit (PCR Biosystems, USA). qRT-PCR assays were performed using a CFX Connect real-time PCR detection system with the CFX Manager software for analysis. The reaction mixtures contained qPCR master mix (New England Biolabs [NEB], USA). Samples without reverse transcriptase or nontemplate controls were included in the qRT-PCR analysis. The relative quantification of each transcript was calculated using the threshold cycle (2−ΔΔCT) method and normalized using the ACT1 gene as an internal control (75 (link)). Sequences of primers used for qRT-PCR are listed in Table 3 .
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from ∼30 mg seedlings in 600 μl of extraction buffer (0.1 M glycine–NaOH, pH 9.0, 100 mM NaCl, 10 mM EDTA, 2% SDS) and extracted with equal volumes of phenol–chloroform (pH 4.3) and chloroform, precipitated with ethanol and resuspended in sterile water. For qRT-PCR assays, 5 μg total RNA was DNase treated (Ambion AM2222, www.thermofisher.com ), precipitated in ethanol and resuspended in sterile water. One microgram of DNase-treated total RNA and random primer was used for the first-strand cDNA reaction (NEB, E6300S, www.neb.com ). qPCRs were done using qPCR Master Mix (NEB, M3003S, www.neb.com ). qPCR reactions were run in a Light Cycler 96 (Roche) real-time PCR machine. At least three biological samples were assessed in each experiment and standard error bars shown. P-values were calculated using unpaired two-tailed Student’s t-test to assess the significance of differences; t-test comparison of samples is shown where considered relevant. For primers, please see Supplementary Table S1 .
6
Quantification of Replication Origins
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Genomic DNA from 1 OD630 of cells was extracted and purified as previously described.82 (link) 3 ng of DNA was used per qPCR reaction (qPCR Master Mix, NEB). PCR was performed using an Applied Biosystems 7500 instrument (software version 2.3). PCR primers are listed in Table 4 . Briefly, qPCR signal for a given origin was first normalized to the signal obtained from the NegV locus (ChrV: 532538-532516).69 (link) This region is located ≈12 kb from ARS521, an origin which has not been detected to be active in several studies according to OriDB (http://cerevisiae.oridb.org/ ) and ≈18 kb from ARS522, a subtelomeric origin of replication activated in late S. As such, the NegV locus is expected to be replicated in late S, and therefore to generally remain unreplicated in a majority of cdc7-4 and cdc7-4 hst3Δ hst4Δ cells 30 min post-release from G1 arrest toward S phase. The NegV-normalized S phase signal was divided by the NegV-normalized signal obtained from alpha factor arrested (G1) cells. Complete replication of an origin is therefore expected to result in a ratio of S phase over G1 signal of 2.
7
RNA Extraction and RT-qPCR Analysis
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Total RNA extraction and purification were performed using TriFast (peqGOLD, USA) and DNase I (Quanta Biosciences, USA) kits according to manufacturer recommendations. One microgram RNA was used for cDNA synthesis using qScript cDNA Synthesis Kit (Quanta Biosciences, Beverly, MA, USA). The RT-qPCR reaction was set as following: 10 µL qPCR Master Mix (NEB, USA), 0.5 µL forward primer (10 µM), 0.5 µL reverse primer (10 µM), 5 µL cDNA and 4 µL nuclease-free water using in the following program: initial denaturation (95°C, 60 s, 1×), denaturation (95°C, 15 s), extension (60°C, 30 s) with 40 cycles and additional melting curve (60°C–95°C, 1×). The primers list is presented in Supplemental Table 2 .
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