Expression of 667 miRNAs was analyzed by a quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) using the Megaplex TaqMan Low‐Density Arrays (TLDAs), version 2.0, system (Applied Biosystems, Foster City, CA, USA). Briefly, 100 ng of total RNA was retro‐transcribed using stem‐loop primers and a pre‐amplification step was added so that the minimum amounts of miRNAs were detected. qPCR assays were performed using GeneAmp System 9700 (Applied Biosystems).
Geneamp system 9700
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The GeneAmp System 9700 is a thermal cycler designed for amplifying DNA sequences using the polymerase chain reaction (PCR) technique. It has a temperature range of 4°C to 99°C and can accommodate standard microplates or tube formats. The system is capable of performing various PCR protocols, including standard, long-range, and real-time PCR.
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Lab products found in correlation
Thermoscript 2 one step rt pcr kit, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sheep blood, by BD (2 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (2 mentions)
Wizard genomic dna purification kit, by Promega (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Titan one tube rt pcr system, by Roche (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (1 mentions)
Mirna assay, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Improm 2, by Promega (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Sangon (1 mentions)
Genegenius, by Syngene (1 mentions)
Genejet miniprep kit, by Thermo Fisher Scientific (1 mentions)
Clonejet pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
High pure pcr product purification kit, by Roche (1 mentions)
Miseq pe300, by Illumina (1 mentions)
Bacterial dna kit, by Omega Bio-Tek (1 mentions)
21 protocols using geneamp system 9700
1
Comprehensive miRNA Expression Analysis
2
First-Strand cDNA Synthesis and RT-PCR Analysis
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First strand cDNA synthesis was performed with 1 μg of total RNA isolated from C2C12 cells at 55°C for 20 min. using the Thermoscript II one-step RT-PCR Kit (Life Technologies, Paisley, UK). cDNA amplification was performed in the same tube using the Gene Amp System 9700 thermocycler (Applied Biosystems, Warrington, UK) followed by heating to 94°C for 5 min. to inactivate the reverse transcriptase. The following PCR conditions were used: 34 cycles each of 30 sec. at 94°C, 30 sec. at 55°C and 60 sec. at 72°C, followed by 10 min. at 72°C. The number of PCR cycles used was optimized to ensure amplification at the exponential phase. Ten-microlitre samples from each RT-PCR reaction were removed and analysed by agarose gel electrophoresis. Bands were stained with ethidium bromide and visualized under ultraviolet (UV) light. The band intensities were quantified using a gel documentation system (Gene Genius, Syngene, UK). The following primers were used: GLUT4-sense (5′-TTG GAG AGA GAG CGT CCA AT-3′) and GLUT4-antisense (5′-CTC AAA GAA GGC CAC AAA GC-3′); β-actin-sense (5′-CAG GAG GAG CAA TGA TCT TGA-3′) and β-actin antisense (5′-ACT ACC TCA TGA AGA TCC TCA-3′). RT-PCR experiment with animal tissues was also performed. Various primers were used as indicated.
3
Extracting and Detecting RNA from Infiltrated Leaves
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Total cellular RNA was extracted by TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) from infiltrated N. benthamiana leaves at 5 days post infiltration (dpi). RT-PCR analysis was carried out using the Titan One Tube RT-PCR System (Roche Molecular Biochemicals, Chicago, IL, USA) as described in the manufacturer's instructions with primer pair AgeTFnt1F/XhoTFntR (0.4 pmol, final concentration). For RT-PCR, 35 cycles were conducted in a GeneAmp®System 9700 (Applied Biosystems, Foster City, CA, USA) with AMV reverse transcriptase for the first strand cDNA synthesis and the Expand High Fidelity enzyme blend (Roche) consisting of Taq DNA polymerase and Tgo DNA polymerase for amplification of cDNA by PCR. The PCR fragments were fractionated on a 1.0% agarose gel.
4
Quantitative Analysis of miRNA Expression
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Quantitative real-time RT-PCR (qRT-PCR) analysis for miRNAs expression was performed using the TaqMan MicroRNA Assay kits (ThermoFisher). Total RNA (100 ng) was reverse transcribed using a looped RT specific primer, dNTPs (100 mM), reverse transcriptase MultiScribe (50 U/μl), 10X buffer, RNase inhibitor (20 U/μl), and 4.16 μl RNase-free water. Retrotranscription reaction (1:15) was mixed with master mix TaqMan (Universal PCR Master Mix, No AmpErase UNG, 2X), and the corresponding specific TaqMan PCR probe. PCR reaction was performed in a GeneAmp System 9700 (Applied Biosystems) as follows: 95°C for 10 min and 40 cycles at 95°C for 15 s and 60°C for 1 min. Tests were normalized using RNU44 as endogenous control.
5
Quantitative Analysis of Vav3 Expression in C2C12 Cells
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First strand cDNA synthesis was performed using 1 µg of total RNA isolated from C2C12 cells at 55℃ for 20 minutes using the Thermoscript II one-step reverse transcription-polymerase chain reaction (RT-PCR) Kit (Invitrogen, Paisly, UK). Amplification of cDNA was carried out in the same tube using the Gene Amp System 9700 thermocycler (Applied Biosystems, Warrington, UK). Heating to 94℃ for 5 minutes inactivated the reverse transcriptase. The following PCR conditions were used: 27 cycles of 30 seconds at 94℃, 30 seconds at 56℃, and 30 seconds at 72℃, followed by 7 minutes at 72℃. The number of PCR cycles used was optimized to ensure amplification at the exponential phase. The 10-µL samples from each RT-PCR product were removed and analyzed by agarose gel electrophoresis. Bands were stained with ethidium bromide and visualized under ultraviolet light. Band intensity quantification was determined by a gel documentation system (Gene Genius, Syngene, UK). The following primers were used: Vav3, 5'-GGCTATCCCGAACACCAATA-3' (sense) and 5'-GAGATGGCTGACTCCACTCC-3' (antisense); and β-actin, 5'-ATT TGG TCG TAT TGG GCG CCT GGT CAC C-3' (sense) and 5' GAA GAT GGT GAT GGG ATT TC-3' (antisense).
6
Quantitative RT-PCR Analysis of miRNAs
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Quantitative RT-PCR analysis of individual microRNAs was performed using MiRNA Assays (Applied Biosystems, Foster City, CA). 100 ng total RNA were reverse transcribed using a looped-RT specific primer, 0.15 μl dNTPs (100 mM), 1.0 μl reverse transcriptase MultiScribe (50 U/μl), 1.5 μl 10X buffer, 0.19 μl RNase inhibitor (20 U/μl) and 4.16 μl RNase-free water. Then, diluted retrotranscription reaction (1:15) was mixed with 10 μl master mix TaqMan (Universal PCR Master Mix, No AmpErase UNG, 2X), 7.67 μl RNase free water, and 1.0 μl PCR probe. PCR reaction was performed in a GeneAmp System 9700 (Applied Biosystems) as follows: 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Tests were normalized using RNU44 and RNU48 as control.
7
RT-PCR Amplification of Glucose, Insulin Receptor
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RT-PCR was performed at 55°C for 20 minutes using the Thermoscript II one-step RT-PCR Kit (Invitrogen). cDNA amplification was performed using a GeneAmpSystem 9700 thermocycler (Applied Biosystems, Warrington, UK). The reverse transcriptase was heat-inactivated in the first step of the PCR reaction (94°C for 5 minutes). The following primers were used for amplification: β-actin, 5′-ATTTGGTCGTATTGGGCGCCTGGTCACC-3′ (sense) and 5′-GAAGATGGTGATGGGATTTC- 3′ (antisense); GLUT3, 5′-CGCAACTCTATGCTTCTAGTCAA- 3′ (sense) and 5′ -ATGCCCAGCTGGTTTAGTGT- 3′ (antisense); and an insulin receptor, 5′-gtactgggagaggcaagcag- 3′ (sense) and 5′-gtgtggtggctgtcacattc- 3′ (antisense). The amplification steps were as follows: 27 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, followed by 7 minutes at 72°C. Ten microliters of product from each RT-PCR reaction were analyzed through agarose gel electrophoresis.
8
Quantification of miR-934 Expression
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We isolated total RNA from MDA-MB-232 and HS578T cell lines using the TRIzol reagent (Invitrogen) and confirmed RNA integrity using an agarose gel. To measure miR-934 expression, we used RT-PCR microRNA assays (Applied Biosystems, Foster City, CA, USA). We used 100 ng of total RNA for reverse transcription, together with 0.15 µL of dNTPs (100 mM), 1.0 µL of reverse transcriptase Mul-tiScribe TM (50 U/µL), 1.5 µL of 10× buffer, 0.19 µL of RNase inhibitor (20 U/µL), and 4.16 µL of RNase-free water. Later, the PCR was performed with 10 µL of TaqMan master mix (Universal PCR Master mix, No AmpErase® UNG, 2×), 7.67 µL of RNase-free water, and 1 µL of taqman probe PCR. The following PCR reaction was performed on an Applied Biosystems GeneAmp System 9700, Foster City, CA, USA: 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 40 cycles of 1 min at 60 °C. The expression of miR-934 was assessed using the Ct (2Ct) method, with U6 as a normalizer.
9
Quantification of miR-944 Expression
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The expression of miR-944 was measured by microRNA assays as implemented by manufacturer (ThermoFisher) and the comparative Ct (2 − ΔΔCt) method using an automatic baseline and a threshold of 0.2 to determine the Ct raw data. Total RNA (100 ng) of cells and tissues was obtained using the Trizol reagent (Invitrogen) and reverse transcribed using the looped-RT specific primer for miR-944, dNTPs (100 mM), reverse transcriptase MultiScribe (50 U/μl), 10X buffer, RNase inhibitor (20 U/μl) and RNase-free water. Then, retrotranscription reaction (1:15) was mixed with 10 μl master mix TaqMan (Universal PCR Master Mix, No AmpErase UNG, 2X), 7.67 μl RNase free water, and 1.0 μl PCR probe. PCR reaction was performed using a GeneAmp System 9700 (Applied Biosystems) as follows: 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. RNU44 was used as a control for normalization of data.
10
qRT-PCR for miRNA Expression Analysis
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cDNA was synthesized from total RNA using TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA). 50 ng of total RNA were used for RT assays using a specific miR-3135b stem loop primer, dNTP (100 mM), MultiScribe reverse transcriptase (50 U/µl), 10X buffer, RNase inhibitor (20 U/µl) and RNase-free water. These reactions were incubated in the thermal cycler at 16 °C for 30 min, 42 °C for 5 min and 85 °C for 5 min. Real-time PCR was carried out in a GeneAmp System 9,700 (Applied Biosystems) using RT product, TaqMan Universal PCR master mix, RNase-free water and miR-3135b probes from the TaqMan MicroRNA Assay protocol kit. Reactions were incubated in a 96-well plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. RNU44 small RNA was used as control for normalizing miRNA data.
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