Total RNA was isolated from cells using RNA‐Bee (Tel‐Test Inc., Friendswood, TX). cDNA was synthesized using M‐MLV reverse transcriptase (Promega, Fitchburg WI) according to the manufacturer's instructions. Quantitative RT‐PCR was performed using the Quantitect SYBRgreen PCR kit (Qiagen, Venlo, The Netherlands) with an iQ5 PCR cycler (BioRad, Hercules, CA). For used primer sets, all spanning at least one intron, see Table 2 . Data were normalized relative to GAPDH expression. Levels of gene expression in differentiation experiments were expressed as fold‐change relative to expression in SaOS‐2 and HDFA cells using the 2−ΔΔCt method. Basal levels of gene expression at beginning of experiments were expressed as fold‐change relative to expression in positive controls (e.g., SaOS‐2 cells, HDFA cells, human endothelial cells, and human monocytes).
Iq5 pcr cycler
Manufactured by Bio-Rad
Sourced in China, United States
The IQ5 PCR cycler is a real-time PCR detection system designed for quantitative PCR (qPCR) analysis. It features a 96-well sample block and a system of optics and detection components that enable the monitoring and quantification of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Minibest universal rna extraction kit, by Takara Bio (2 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Rna bee, by Tel-Test (1 mentions)
Primescript rt master mix perfect real time kit, by Takara Bio (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rna isolation kit, by Beyotime (1 mentions)
Sybr green pro taq hs, by Accurate Biology (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taqtm 2 kit, by Takara Bio (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
5 protocols using iq5 pcr cycler
1
Quantitative RT-PCR Analysis of Gene Expression
2
Quantifying Gene Expression in Vertebrae
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The levels of expression of mRNAs encoding CTSK and Runx2 were evaluated by RT-PCR. Total RNAs from L5 vertebrae were extracted using the protocol of the Takara MiniBEST Universal RNA Extraction Kit (catalogue no. 9767, Takara, Japan). Sixty microliter amounts of reaction solution (10 μL of solution can contain up to 500 ng of RNA) were subjected to reverse transcription using the PrimeScriptTM RT Master Mix (Perfect Real Time) kit (catalogue no. RR036A, Takara). Real-time analysis was performed with the aid of a Bio-Rad iQ5 PCR cycler using a SYBRR Premix Ex TaqTMII (TilRNaseH Plus) kit (catalogue no. RR820A, Takara). β-actin served as the internal standard. 2 ul cDNA was amplified with the special primer as follow: 5′-ACTCTGAAGACGCTTACCCG-3′ and 5′-CCTTTGCCGTGGCGTTATAC-3′ for CTSK; 5′-CAGACCAGCAGCACTCCATA-3′ and 5′-GCTTCCATCAGCGTCAACAC-3′ for Runx2; 5′-GGAGATTACTGCCCTGGCTCCTA-3′ and 5′-GACTCATCGTACTCCTGCTTGCTG-3′ for β-actin. Relative gene expression levels were quantified using the 2−DΔCt method.
3
qRT-PCR Analysis of BMSC Gene Expression
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BMSCs were harvested and total RNA was extracted using RNA easy total RNA kit (Takara Bio, Inc.) according to the manufacturer's instructions. RNA was reverse-transcribed into cDNA at 42°C for 2 h using the PrimeScript RT Master Mix kit (Perfect Real Time, cat. no. RR036A; Takara Bio, Inc.). The primers are listed in Table I . RT-qPCR was performed using the SYBRR Premix Ex TaqTMII kit (TilRNaseH Plus, cat. no. RR820A; Takara Bio, Inc.) on an iQ5 PCR cycler (Bio-Rad Laboratories, Inc.). The PCR thermocycling conditions were as follows: 52°C for 2 min and 95°C for 5 min, followed by 45 cycles at 95°C for 15 sec and 58°C for 60 sec. The relative mRNA expression levels were calculated using the 2−ΔΔCq method (31 (link)). Expression was normalized to β-actin.
4
Quantification of Gene Expression in Mouse Colon and Hypothalamus
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Total RNA was extracted from the colon and hypothalamus samples using RNA isolation kit (R0027, Beyotime, Beijing, China) and then reverse transcribed to cDNA with Reverse Transcription Kit (K1622, Thermo Scientific, Waltham, MA, USA). Gene expression was quantified by qPCR using SYBR Green Pro Taq HS (AG11701, Accurate Biology, Changsha, Hunan, China) in an iQ5 PCR cycler (Bio-Rad, Hercules, CA, USA) with specific primers. All the results were analyzed using the −ΔΔCt method and normalized to the reference gene Actb. The primer sequences are listed in Table 1 .
5
Quantifying Gene Expression in HGC-27 Cells
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HGC-27 cells (5 × 10 6 ) treated by Kangai injection for 48 hours were harvested. Total RNA was extracted using the MiniBEST Universal RNA Extraction Kit (Takara, Japan). RNA purity was assessed by measuring the A260/A280 ratio using a NanoDrop (ThermoScientific, USA) and RNA quality checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Total extracted RNA (500 ng) was subjected to reverse transcription using the PrimeScript RT Master Mix kit (Takara, Japan). RT-qPCR was performed using the SYBR Premix Ex TaqTMII kit (Takara, Japan) on a Bio-Rad iQ5 PCR cycler (USA). β-actin served as the internal reference. The parameters were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cy-cles of 95 °C for 15 s and 60 °C for 60 s. All experiments were performed in triplicate. Relative levels of genes or RNAs were normalized to β-actin using the 2 -ΔΔCt method (Table 1).
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