Total RNAs from tissues or cells were isolated by a TRIzol Reagent (Invitrogen, Waltham, MA, USA), followed by the reverse transcription of extracted RNAs through a miScript II RT kit (QIAGEN, Hilden, Germany) in a fluorescence thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The expression of relative genes was measured by a SYBR Green PCR reagent kit (Applied Biosystems) with the following primer sets on an ABI ViiA7 Real-Time PCR System. The relative expression of target genes was normalized to β-actin. The biological information and primer sequences are shown in Table 2 and supplementary Figure 1 , respectively.
Fluorescence thermal cycler
Manufactured by Bio-Rad
Sourced in United States
The Fluorescence thermal cycler is a laboratory instrument designed for thermal cycling applications. It utilizes fluorescence detection for real-time monitoring of nucleic acid amplification, such as in quantitative PCR (qPCR) and reverse transcription PCR (RT-PCR) experiments. The core function of the Fluorescence thermal cycler is to precisely control the temperature of samples during thermal cycling protocols.
Automatically generated - may contain errors
Lab products found in correlation
Miscript 2 rt kit, by Qiagen (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Miscript sybr green pcr kit, by Qiagen (3 mentions)
Mirneasy mini kit, by Qiagen (3 mentions)
Sybr green pcr reagent kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Abi 7300 qpcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnx plus reagent, by CinnaGen (1 mentions)
Sybr green 1 real time pcr kit, by Vazyme (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using fluorescence thermal cycler
1
Quantitative Gene Expression Analysis
2
RNA Isolation and miRNA Detection
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Total RNA was isolated from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Serum miRNAs were extracted from 400 µl of serum using the miRNeasy Mini kit and reversely transcribed using the miScript II RT kit (both from Qiagen, Hilden, Germany). The levels of miRNAs were detected using the miScript SYBR-Green PCR kit (Qiagen) in a Bio-Rad fluorescence thermal cycler (Bio-Rad, Hercules, CA, USA). The relative expression levels of the miRNAs were normalized to that of U6.
3
Quantitative PCR for Gene Expression
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Total RNA from cells was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was produced by reverse transcription using the SuperScript® III RT-PCR kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Inc.). The amplification of fluorescence signals was detected by a fluorescence thermal cycler (Bio-Rad Laboratories, Inc., United States). RT-qPCR was performed (Novoprotein, Shanghai, China) using the following primers: RPS3 sense, 5′-GCGAGTTACACCAACCAGGA-3′ and antisense, 5′-ATGAACCGCAGCACACCATA-3′; β-actin sense, 5′-AGCAGCATCGCCCCAAAGTT-3′ and antisense, 5′-GGGCACGAAGGCTCATCATT-3′. B -Actin was set as an internal control for cellular mRNAs. The parameters for PCR quantification were as follows: 2 min at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C. The results of qPCR were defined using the quantification cycle (Cq), and 2–ΔΔCq was used to calculate the relative expression levels (Livak and Schmittgen, 2001 (link)).
4
Exosomal miRNA and mRNA Quantification
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Total RNA from cells and exosomes was extracted using an miRNeasy Mini Kit (Qiagen, Inc., Hilden, Germany). miRNAs and mRNAs were reverse transcribed using the miScript II RT Kit (Qiagen, Inc.) and detected by miScript SYBR Green PCR Kit (Qiagen, Inc.) according to the manufacturer’s protocols. The amplification of fluorescence signals was detected by a fluorescence thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RNU6B and β-actin were set as internal controls for cellular miRNAs and mRNAs, respectively. The relative expression levels of exosomal miRNA were normalized to miR-16-5p. The 2−ΔΔCq method was used for miRNAs and mRNAs quantification (27 (link)); miScript primers for miRNAs and RNU6B (cat. no. 218300) were obtained from Qiagen, Inc. The primers sequences for mRNAs detection and the thermocycling conditions applied are listed in Table I ; experiments were conducted ≥3 times.
5
Quantification of miRNA Expression
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Total RNAs from tissues or cells were isolated using RNX™-Plus Reagent (Cinnagen) and cDNA was synthesized using the PrimeScript™ RT Reagent Kit (Takara) according to the manufacturer's instructions. qPCR was performed using a SYBR Premix ExTaq™ kit (Takara), with the following primer sets on the ABI 7300 qPCR system (Applied Biosystems). β-actin was used to normalize the relative expression of the target genes. miRNAs were detected through a miScript II RT kit (Qiagen) in a fluorescence thermal cycler (Bio-Rad Laboratories, Inc.). The primers for miR-188-5p and the reference gene U6 were purchased from Novland Biopharm. The thermocycling condition were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1min, followed by a hold at 4°C. The relative expression ratio of miR-188-5p was quantified using the 2−ΔΔCq method (10 (link)). The relative expression of miR-188-5p was normalized to U6. The primer sequences are listed in Table I .
6
Exosomal and Serum miRNA Profiling
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Total RNA from the tissues and cells was isolated using TRIzol Reagent (Invitrogen). Exosomal and serum miRNAs were extracted from 400 μl of serum by using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) and reversely transcribed using the miScript II RT Kit (Qiagen). Quantitative RT–PCR (qRT–PCR) assays were performed using the miScript SYBR Green PCR Kit (Qiagen) and the Bio-Rad fluorescence thermal cycler (Bio-Rad Laboratories, lnc., Hercules, CA, USA) for quantitative miRNA detection. The relative expression levels of the miRNAs in tissue and cells were normalised to U6 and those in the serum and exosomes were normalised to the insert control according to the manufacturer's protocol.
7
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from the cells using TRIzol® reagent (Life Technologies) according to the manufacturer’s instructions, and an equal amount of RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis kit (Fermentas, Glen Burnie, MD, USA). RT-qPCR was performed to detect the changes in mRNA expression using the SYBR-Green I Real-Time PCR kit (Vazyme Biotech Co., Ltd., Nanjing, China) and the Bio-Rad fluorescence thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). The relative mRNA expression was normalized to the insert control gene, β-actin, according to the manufacturer’s instructions. The primers used in the present study were produced by Invitrogen (Life Technologies). All primer sequences and RT-qPCR conditions are listed in Table I .
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