Using an ABI PRISM 7500 instrument, real-time PCR was performed with SYBR Premix Ex Taq II (TaKaRa Bio, Inc., Dalian, China) according to manufacturer's instructions. To detect downstream genes possibly regulated by IgG, common metastatic genes MTA1, CD44, E-cadherin, MMP9, MMP2 and Intergrin β1 were examined following siRNA interference (see “RNA interference”). The primers used for real-time PCR are listed in Table S3 . Amplification of β-actin was used for normalization. The relative expressions of each gene in the tested cell types were determined with the method of 2−ΔΔCt[36] (link).
Prism 7500
Manufactured by Takara Bio
Sourced in China, Japan
The PRISM 7500 is a real-time PCR system designed for quantitative gene expression analysis. It features a sensitive optical detection system and compatible software for data collection and analysis. The PRISM 7500 supports a range of sample volumes and can accommodate multiple reaction formats.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Superscript 2 rt, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr kit, by Takara Bio (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt master mix perfect real time kit, by Takara Bio (1 mentions)
Multisource rna miniprep kit, by Corning (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq tli rnaseh plus, by Takara Bio (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
5 protocols using prism 7500
1
Investigating Metastatic Gene Expression
2
Quantitative RT-PCR Analysis of Human Genes
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Total RNA was extracted using Trizol Reagent treated with DNase I and 2 μg RNA was reverse-transcribed using Superscript II RT following the manufacturer's instructions (Life technologies, USA). Primers for qRT-PCR were designed using Primer premier software 5.0. Human GAPDH primers used as an internal control were 5′-GAAGGTGAAGGTCGGAGTC-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse). Human RPS27a primers were 5′-AGAAGAAGTCTTAC ACCACTCCC-3′ (forward) and 5′-TGCCATAAACACCC CAGC-3′ (reverse). Human STAT3 primers were 5′-CAGT TTCTTCAGAGCAGGTA-3′ (forward) and 5′-CTTGACT CTTGAGGGTTTT-3′ (reverse). The qRT-PCR products were 226 bp, 158 bp and 286 bp, respectively. The qRT-PCR was performed with SYBR Green PCR kit (Takara, Japan) following the manufacturer's instructions on the ABI PRISM7500 real-time PCR system. Thermal cycling conditions were 95°C for 5 min, followed by 40 cycles of 5 s at 95°C, and 34 s at 60°C. The qRT-PCR reactions were performed in a total volume of 20 μl, containing 2 μl of sample cDNA, 0.2 μM of each primer.
3
Quantification of Apoptosis Markers
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Samples of heart tissue (200 mg) were processed by RT-qPCR. Following the manufacturer's instructions, we extracted apoptosis-associated RNAs such as Bax, Bcl-2, and caspase-3, which were determined by RT-qPCR for relative levels in CME. The ABI PRISM-7500 machine (CA, USA) was used to perform RT-qPCR using the TB Green® Premix Ex Taq™ II (TaKaRa, Japan). The specific primer sequences used in this study were listed in Table 1.
4
Quantification of Gene Expression in Lung Tissue
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Lung tissue total RNA was isolated using a Multisource RNA Miniprep kit (Axygen, USA). The total RNA was reverse transcribed into cDNA using PrimeScript™ RT Master Mix (Perfect Real Time) Kit (Takara, China). qPCR was performed using an ABI Prism 7500 instrument following the SYBR-Green reagent protocol (Takara, China). Data were analyzed as fold change by the 2−ΔΔCt method. Primers used in this study are listed below: β-actin forward: 5′-gccaaccgtgaaaagatg-3′, β-actin reverse: 5′-tgccagtggtacgaccag-3′; Rras2 forward: 5′-gaccatggcttttgcttgct-3′, Rras2 reverse: 5′-tagcggggacattgaacgtg-3′; Ttll12 forward: 5′-gcatccagagagttcgcaga-3′, Ttll12 reverse: 5′-gggtctcgggtgtaacacag-3′; Nog forward: 5′-tgtacgcgtggaacgaccta-3′, Nog reverse: 5′-ggcttacacaccatgccctc-3′; Vangl2 forward: 5′-tgatccccgattgcttggtc-3′, Vangl2 reverse: 5′-ccagaccactcggctgttt-3′; Gli3 forward: 5′-atcagccctgctttgagctt-3′, Gli3 reverse: 5′-gatgggtctctgcgttggaa-3′; Trps1 forward: 5′-gagcagcagaggatctggag-3′, Trps1 reverse: 5′-cctagtctgctccccgtttg-3′; Tc1 forward: 5′- ctcttcgagtaagcccgtcc-3′, Tc1 reverse: 5′-gggctcgagttttctcctcc-3’.
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen) and first-strand cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). RT-qPCR was performed in an ABI PRISM 7500 Fast Real-Time PCR system using SYBR ® Premix Ex Taq™ (Tli RNase H Plus) according to the manufacturer's protocol (Takara, Otsu, Japan). Table II lists the primer sequences used.
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