Primary normal and tumour tissues (obtained from KMIO) were processed using motorized homogenizer, the snap frozen tissue (~100 mg) was ground and total RNA was isolated using Tri-reagent (Sigma Aldrich, St Louis, MO, USA) according to manufacturer’s protocol. cDNA was synthesized from 2 μg of total RNA using Gene-Amp RNA PCR cDNA synthesis kit (Applied Biosystems, Carlsbad, CA, USA). Primers were designed using Primer3 online tool. GAPDH and β2-microglobulin were used as normalizing controls. Sequence of primers used is provided in S1 Fig . Real Time PCR was performed according to manufacturer’s protocol using DyNAmo SYBR Green qPCR Kit (Finnzymes, Vantaa, Finland) with ROX as passive reference dye using Applied Biosystem’s 7900 HT Real Time PCR system. The following PCR program was used for all real time PCR based experiments: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 5 minutes.
Dynamo sybr green qpcr kit
Manufactured by Thermo Fisher Scientific
Sourced in Finland, United States
The DyNAmo SYBR Green qPCR Kit is a reagent kit designed for quantitative real-time PCR (qPCR) analysis. It contains a SYBR Green-based master mix and is used for the detection and quantification of DNA sequences.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (8 mentions)
Dnase 1, by Thermo Fisher Scientific (7 mentions)
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Pcr blunt vector, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Dna engine opticon 1, by Bio-Rad (3 mentions)
Dnase 1 kit, by Thermo Fisher Scientific (3 mentions)
Lightcycler probe design v2, by Roche (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Gene amp rna pcr cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Merck Group (2 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (2 mentions)
Fx96 real time pcr detection system, by Bio-Rad (2 mentions)
Trizol extraction method, by Thermo Fisher Scientific (2 mentions)
Dynamo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol method, by Thermo Fisher Scientific (2 mentions)
Geneamp rna pcr kit, by Thermo Fisher Scientific (2 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (2 mentions)
Rna stat 60 reagent, by Tel-Test (2 mentions)
43 protocols using dynamo sybr green qpcr kit
1
RNA Isolation and qPCR Workflow for Tissue Samples
2
mRNA Quantification of mDia1 and mDia2 in Mouse Oocytes
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The mRNA levels of mDia1 and mDia2 in mouse oocytes were determined by using real-time quantitative PCR. Total RNA was extracted from 50 oocytes using a Dynabeads mRNA DIRECT Kit (Life Technologies, Foster City, CA, USA). First-strand cDNA was generated using a cDNA Synthesis Kit (Takara, Kyoto, Japan) and oligo(dT) 12–18 primers. The PCR primers used to amplify mDia1 and mDia2 are listed in Table 1 . Real-time PCR was performed with SYBR Green in a final reaction volume of 20 μl (DyNAmo SYBR Green qPCR Kit; Finnzymes, Vantaa, Finland). PCR conditions were as follows: initial denaturation at 94°C for 10 min, followed by 39 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s, and a final extension at 72°C for 5 min. Gene expression was normalized to the level of GAPDH mRNA and quantified by using the ΔΔCT method[39 (link)]. Experiments were conducted in triplicate.
3
Quantitative Real-Time PCR Assay Development
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Real-time PCR was carried out using a DyNAmo SYBR Green qPCR kit (Finnzymes). The PCR mixture, which contained 20 pmol of forward and reverse primers and 2 μl of cDNA, was subjected to amplification with a DNA Engine Opticon 1 (MJ Research). The cycles were set at 95°C for 10 min for preheating, followed by 40 cycles at 94°C for 15 s, at 55°C for 30 s and at 72°C for 30 s. The amplicons were detected directly by measuring the increase in fluorescence caused by the binding of the SYBR Green I dye to gene-specific and amplified double-strand DNA using a DNA Engine Opticon 1. Following the completion of the PCR reaction, the temperature was raised from the annealing temperature to 95°C for melting curve analysis. The expression level was calculated by the 2−ΔΔCt method and compared with the relative expression. Primer sequences are shown in Table 1 .
4
RNA Extraction and qRT-PCR Analysis
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Total RNA from cells, explants and snap-frozen ovarian tumors were prepared using TRIzol extraction method (Invitrogen, Carlsbad, CA). The quantity and quality of isolated RNA was determined by NanoDrop (Thermo Fisher Scientific Inc., Waltham, MA, USA) and gel electrophoresis. Before the reverse transcription (RT) reaction 1 μg of total RNA was incubated for 30 min with DNase I (Invitrogen, Carlsbad, CA) at room temperature. The RT reaction was performed with DyNAmo ™ cDNA Synthesis Kit (Finnzymes, Espo. Finland) at 37 °C for 1 h in 20 μl. Quantification of investigated genes was performed with FX96™ Real-Time PCR Detection System, Bio Rad using DyNAmo SYBR Green qPCR kit (Finnzymes). Reaction conditions were: initial denaturation at 95 °C for 10 min followed by 40 amplification cycles at 95 °C for 15 s, 56–60 °C at 45 s and 70 °C at 45 s. At the end of the PCR reaction, melting curve was determined to ensure single product amplification. Amplification products were separated on 1.8% agarose gel and stained with ethidium bromide. Expression levels were normalized to the housekeeping gene peptidylprolyl isomerase (PPIA). The primer sequences and expected product sizes are shown in Supplementary Table S1.
5
Quantitative RT-PCR Analysis of RNA Expression
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Total RNA was isolated from FLS using TriFast (Peqlab, Erlangen, Germany) and reverse transcribed with random hexamers (Finnzymes, Espoo, Finland). qRT-PCR was performed with a DyNAmo SYBR Green qPCR kit (Finnzymes) under the following thermal conditions: 95°C for 7 min. and 40 cycles of 95°C for 20 sec., 60°C for 30 sec. and 72°C for 15 sec. GAPDH was used as a normalizing control. The gene sequence of GAPDH (NM_002046.3) was derived from Ensembl Human Genome Browser. Forward (5′-CGG GGC TCT CCA GAA CAT C-3′) and reverse (5′-CTC CGA CGC CTG CTT CAC-3′) oligonucleotide primers were designed using the primer three programme and purchased from Eurofins-MWG Operon (Ebersberg, Germany). Oligonucleotide primers specific for MMP-2 (forward: 5′-GCG ACA AGA AGT ATG GCT TC-3′ and reverse: 5′-TGC CAA GGT CAA TGT CAG GA-3′) and MMP-14 (forward: 5′-CAA CAC TGC CTA CGA GAG GA-3′ and reverse: 5′-GTT CTA CCT TCA GCT TCT GG-3′) were derived from Konttinen et al. [25 (link)].
6
Quantitative RT-PCR Analysis of BAT
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Total RNA was isolated from interscapular BAT obtained from 10- to 12-week-old male mice in each condition, and cDNA were produced from 1 μg of total RNA according to methods reported in a previous study (32 (link)). For real-time quantitative PCR, 1 μl of the cDNA product was added to 20-μl reaction volume containing 10 μl DyNAmo SyBR Green qPCR Kit (Finnzymes) and 1 μl of 12.5 pM primers (forward and reverse). For each sample, a parallel reaction was set up with acidic ribosomal phosphoprotein P0 (Arbp) as an endogenous control. The reactions were run in a DNA Engine Opticon System (MJ, Japan). Each reaction was performed in duplicate. In real-time PCR analysis, the following primers were used: UCP1, forward, 5′-GTGAAGGTCAGAATGCAAGC-3′ and reverse, 5′-AGGGCCCCCTTCATGAGGTC-3′; α2A-AR, forward, 5′-CGCTCAAAGCTCCCCAAAAC-3′ and reverse, 5′-GCTTCAGGTTGTACTCGATGGC-3′; β3-AR, forward, 5′-GGACCTGCACTACCACCTGT-3′ and reverse, 5′-CATGAGGCCTCTTCTTGGAG-3′; Arbp, forward, 5′-ATAACCCTGAAGTGCTCGACAT-3′ and reverse, 5′-GGGAAGGTGTACTCAGTCTCCA-3′.
7
Quantifying Relative mRNA Levels
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To compare the relative mRNA levels in samples, cDNA synthesized from total RNA was quantified using real-time PCR and a DyNAmo SYBR Green qPCR Kit (Finnzymes, Espoo, Finland). The mRNA level of each gene was normalized to that of β-actin and defined as 2-ΔΔCt, where Ct = the threshold cycle for target amplification, ΔCt = Cttarget gene – Ctinternal reference (β-actin), and ΔΔCt = ΔCtsample – ΔCtcalibrator.
8
Quantitative gene expression analysis
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Total RNA was isolated with the Nucleospin RNA II isolation kit (Macherey-Nagel EURL, Fr). The absence of DNA was verified by PCR for G3PDH. One μg of RNA was subjected to ABI High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR with SYBR Green was performed with DyNAmo SYBR Green qPCR Kit (Finnzymes, Oy, Finland), using the StepOnePlus™ System (Applied Biosystems), at 95°C for 3 min followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, 72°C for 60 s. PCR reactions were performed using the following primer pairs (synthesized by Eurofins Genomics): c-MYC : forward CAC CGA GTC GTA GTC GAG GT and reverse TTT CGG GTA GTG GAA AAC CA SOX-2 : forward AGG AGC CTT CCT TTT CCA GA and reverse CGA CGA GAG CTC CTA CCA AC, ALDH1A1 : forward TCC TCC TCA GTT GCA GGA TT and reverse GCA CGC CAG ACT TAC CTG TC, KLF4 : forward GTC AGT TCA TCT GAG CGG G and reverse AGA GTT CCC ATC TCA AGG CA. The number of cycles used was optimized to fall within the linear range of PCR amplification. Changes were normalized according to cyclophilin A expression (forward: ATG GTC AAC CCC ACC GTG T and reverse: TTC TGC TGT CTT TGG AAC TTT GTC).
9
Quantitative 16S rRNA Profiling
10
RNA Isolation, cDNA Synthesis, and Real-Time PCR Analysis
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Total RNA was isolated from leaves by following phenol chloroform method [28] (link). Total RNA was reverse transcribed to generate cDNA by using Revert Aid Reverse Transcriptase (MMLV-RT; MBI Fermentas, Hanover, MD, USA) using oligo (dT) primers (Table S1 in File S1 ) following manufacturer’s instructions. Real-time PCR was performed in the presence of SYBR-green fluorescence dye (DyNAmo SYBR-Green qPCR Kit FiNNZYMES, Finland) using equal amount of cDNA. The critical threshold cycle (Ct) values were normalized using Ct obtained for elongation factor-A (ELF-A) in respective samples and relative expression was calculated [29] (link).
The downstream target genes of AtDREB2A, AtHB7 and AtABF3 were studied by RT-PCR analysis. The target gene sequences were obtained from peanut ESTs (http://www.ncbi.nlm.nih.gov ) for designing primers (Table S1 in File S1 ).
The downstream target genes of AtDREB2A, AtHB7 and AtABF3 were studied by RT-PCR analysis. The target gene sequences were obtained from peanut ESTs (
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