Real-time qPCR reactions for the quantification of libraries were conducted using the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each reaction contained 1X GeneAmp PCR buffer I (Applied Biosystems, Waltham, MA, USA), 0.05 U/μL Taq M hot-start DNA polymerase (AlkorBio, St.-Petersburg, Russia), 2.5 mM MgCl2, 250 μM of each dNTP, 1-x solution of EvaGreen dye (Biotium, Fremont, CA, USA) and 200 nM of each primer (5′-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGT-3′ and 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3′). The volume of PCR reaction was 20 μL, and 2 μL of 1:10000 diluted DNA library was added per reaction. Libraries were quantified against the sequential dilutions of two 2 nM libraries, which were prepared earlier from FFPE materials. The dilution process and qPCR measurements were repeated twice for each library, and the mean value of two measurements was taken as the final result. Fragment sizes of amplified libraries were measured with 5200 Fragment Analyzer System (Agilent Technologies, Santa Clara, CA, USA).
Evagreen dye
Manufactured by Biotium
Sourced in United States, China, Switzerland, Germany, United Kingdom, Lithuania
EvaGreen is a double-stranded DNA-binding dye. It is a sensitive fluorescent dye that can be used for quantitative real-time PCR (qPCR) and other nucleic acid detection applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (24 mentions)
Trizol, by Thermo Fisher Scientific (10 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (7 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (7 mentions)
Cfx96 real time system, by Bio-Rad (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (6 mentions)
Bst 2.0 warmstart dna polymerase, by New England Biolabs (5 mentions)
M mlv reverse transcriptase, by Promega (5 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Mgso4, by New England Biolabs (4 mentions)
Betaine, by Merck Group (4 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (4 mentions)
Rotor gene 6000, by Qiagen (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Isothermal master mix, by OptiGene (4 mentions)
Lightcycler 96 system, by Roche (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
142 protocols using evagreen dye
1
Quantification of DNA Libraries by qPCR
2
Quantifying Uracil Content in DNA
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In order to quantify the uracil content of DNA, a real-time quantitative PCR-based assay was used38 (link). Genomic DNA was isolated and digested with BamHI. DNA fragments of 5 kb were purified from gel. Real-time PCR was performed on a Mx3000P qPCR System (Agilent Technologies) using EvaGreen dye (Biotium) and PfuTurbo Hotstart DNA polymerase (Stratagene) and Mytaq Hotstart DNA polymerase (Bioline). A segment with 1017 base length defined by the primers (Table S41 ) was amplified during the PCR reaction. Two-fold dilution series were prepared from the DNA samples. Three parallel strains were used for each mutation in the experiments.
3
Real-time PCR with In-house SYBR Green and EvaGreen Mixes
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Complementary DNA (cDNA) was diluted (1:10) times and used as a template for Real-time PCR. ABI Power SYBR Green PCR master mix was used as commercial reagent. 2x in-house SYBR Green I and 2x in-house EvaGreen mix was prepared as described below. SYBR Green I (Lonza Cat no. 50513) or EvaGreen dye (Biotium Cat no. 31000), dNTP mix (LAROVA Cat no. DMIX10_100ML) were used to prepare mastermix. ABI 384 well plate (cat no. AB 1384) was used to set up real time PCR. 5µl of mastermix was added to each well. 1µl of (1:10) diluted cDNA was added as template. Total reaction volume was 6µl. ABI 7900 HT machine was used to perform Real-time PCR. Detailed composition of SYBR Green I and EvaGreen PCR mastermix is given below:
| 2X In-house SYBR Green I mix composition | 2X In-house EvaGreen mix composition |
|---|---|
2X In-house buffer – 1000 µl 10 mM dNTP – 25 µl 10000X SYBR Green I – 0.1 µl Hotstart Taq – 10 µl | 2X In-house buffer – 1000 µl 10 mM dNTP – 25 µl 20X Evagreen – 100 µl Hotstart Taq – 10 µl |
4
Betaine and HNB-Based Molecular Assay
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Betaine and hydroxy naphthol blue (HNB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The dNTPs were purchased from Promega (Madison, WI, USA). EvaGreen dye was obtained from Biotium (Hayward, CA, USA). Bst DNA polymerase was bought from Summerbio (Beijing, China). An AMV reverse transcriptase kit was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). Plasmid extraction kits (Plasmid Mini Kit I) were purchased from Omega (Norcross, GA, USA). The in vitro transcription T7 kit for RNA synthesis was provided by TaKaRa (Dalian, China).
5
Transcript Analysis of Developing Soybean Seeds
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Parental lines FUKU and MUT were grown in a greenhouse (February to June 2018) under a long-day photoperiod (14.5 h/9.5 h) until the end of March. Developing seed was collected randomly from several plants at 40, 55, and 70 days after flowering (DAF). Total RNA was isolated from developing seed (50–100 mg) with a Total RNA Extraction Kit Mini Plant (SciTrove, Tokyo, Japan) with additional rDNase I (Takara Bio Inc., Shiga, Japan) treatment. cDNA was synthesized from 1 μg total RNA with ReverTra Ace (Toyobo, Osaka, Japan). A 5-μL aliquot (approx. 25 ng) of cDNA was used as a template. Quantitative real-time PCR was conducted as follows in a LightCycler 96 system: 95°C for 5 min for activation of enzyme, 95°C for 15 s, 60°C for 15 s, 72°C for 20 s, for a total of 40 amplification cycles. EvaGreen Dye (20× in water; Biotium, Inc., Fremont, CA, USA) was used as the fluorescent dye, and dNTPs (Takara), homemade recombinant Taq polymerase, the PCR buffer mentioned in a previous study (Yamagata et al. 2018 (link)), and 1 pmol of each gene-specific primer were used to evaluate transcript levels of target genes. Expression relative to GmACT2/7 (Glyma.19G147900), the internal reference gene, was calculated by using the 2-ΔΔCt method (Schmittgen and Livak 2008 (link)). Data from four technical replicates were analyzed. The primers used are listed in Supplemental Table 1 .
6
Measuring ASO-RNA Duplex Stability
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ASO and target RNA oligonucleotides were diluted in a 100 mM NaCl/100 mM sodium phosphate/0.1 mM ethylenediaminetetraacetic acid (EDTA) buffer to a 10 ng/μl concentration, and mixed in a 1:1 ratio. EvaGreen dye (Biotium) was prepared in 1× Tris EDTA (TE) buffer (final concentration of 3 μM), mixed in 1:1 ratio with the duplex solution and transferred to a white 384-well plate (10 μl per well). The plate was sealed with adhesive tape, centrifuged briefly at 2500 rpm and placed into a Roche LightCycler 480 II. The melting temperature (Tm) analysis was carried out using the protocol described in Supplementary Table S4, which measures fluorescence at 465–510 nm with both ‘Max Peaks (two or less)’ and ‘SYBR Green I format’ options turned on. This produced melt peaks (based on the first negative derivative of the melt curves) and Tm values for each sample. The average Tm of an ASO/RNA duplex was determined from a minimum of four replicates per experiment, and the experiment was repeated a minimum of three times. The list of oligonucleotide sequences corresponding to predicted off-target binding sites is shown in Supplementary Table S5.
7
Technical Validation of RNA-seq DEGs
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As RNA-seq data was generated under a non-biological replicated design, technical validation was performed in order to verify analysis results. We performed qRT-PCR experiments with 6 RBC-breed samples of brain tissue (n = 3, male and female, respectively) for 5 significantly detected DEGs- CHD1, NIPBL, VIP, HXC4, and MYP0. Total RNA was isolated from 4 different tissues using the TRIzol reagent (Invitrogen Carlsbad, CA, USA) according to manufacturer's instructions. We used Applied Biosystems Step-One Real-Time PCR system, EvaGreen dye (Biotium, Hayward, CA, USA) with β-actin (ACTB) as the control gene in order to determine relative gene expression (ΔCt). Primers for 5 DEGs are listed in S3 Appendix . In addition, a two-group t-test was employed to determine gene significance.
8
Improved RT-qPCR Detection Sensitivity
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Example 8
The M160 (
Reactions (20 μl) contained 50 mM Tris, pH 8.7, 75 mM KCl, 4 mM MgCl2, 0.3 mM dNTPs, 0.04 mg/ml human serum albumin, 0.2 M trehalose, 0.225× EvaGreen dye (Biotium), the indicated number of copies of MS2 phage RNA, 0.3 μM forward and reverse primer (25 nucleotides each in size), and 300 ng polymerase. The amplicon was 531 bp in size and corresponded to position 184 to 714 of the MS2 genome (GenBank Acc. No. V00642.1; SEQ ID NO:23). Reactions were thermal cycled in a StepOnePlus (Thermo Fisher) as follows: 94° C. 30 sec (×1), 94° C. 3 sec, 64° C. 1 minute (×40). Compared with M160 alone (
9
Real-time mCOP-PCR for SMN1 and SMN2 Exon 7
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Real-time mCOP-PCR SMN1 and SMN2 exon 7 amplification was performed using the LightCycler® 96 system (Roche Applied Science, Mannheim, Germany). An aliquot of pre-amplified PCR product was added to the reaction mixture with DNA polymerase KOD FX Neo (TOYOBO) and EvaGreen® Dye (Biotium, Hayward, CA, USA). The primer set for SMN1-specific amplification consisted of R111 and SMN1-COP (5′-TGT CTG AAA CC-3′) [18 (link),21 (link)]. The PCR conditions for the reaction mixture of 50 µL were: (1) initial denaturation at 94 °C for 7 min; (2) 20 cycles for SMN1 denaturation at 94 °C for 1 min, annealing at 37 °C for 1 min, and extension at 72 °C for 1 min; and (3) melting analysis. Fluorescence signals were detected at the end of each extension procedure.
10
SNP Marker Development and Allele Determination
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High Resolution Melting curve (HRM) markers were developed from SNPs identified from GBS. Primers were developed using the IDT PrimerQuest (idtdna.com/Primerquest). DNA was extracted using a NaOH rapid DNA extraction method (Lee et al., 2017 (link)). The 5 μl PCR reactions were mixed with 2x AccuStart II PCR SuperMix (Quantabio, Beverly, MA), 0.5 μM of each primer, and 20x EvaGreen Dye (Biotium, Hayward, CA) and run as follows: (95 °C @ 60s) + 40 × ((94 °C @ 5s) + (Tm @ 10s) + (72 °C @ 15s)) + (72 °C for 60s), where Tm is the annealing temperature. For allele determination, melting curve analysis was performed by scanning the PCR product in a LightCycler 480 Instrument II (Roche, Pleasanton, CA).
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