A small piece of frozen tissue was subjected to TRIzol® (Life Technologies, CA, USA) for isolation of RNA, as per the manufacturer's protocol. 2 µg of RNA was treated with DNase I (ThermoFisher Scientific, MA, USA) and reverse transcribed to CDNA using RevertAid H Minus Reverse Transcriptase and oligo-dT primers (ThermoFisher Scientific). Real-time quantitative reverse-transcription PCR (qRT-PCR; qPCR) was performed using the ABI 7900HT Fast RT-PCR system (Applied Biosystems), Sybr Green (ThermoFisher Scientific) and primers designed to specifically hybridize to the corresponding genes of interest. Relative gene expression was calculated for WT and Fabry samples using expression standard curves and normalization to endogenous controls (β-actin and/or glyceraldehyde 3-phosphate dehydrogenase [GAPDH]). Then, expression fold-changes of Fabry samples were calculated over the WT samples.
Abi 7900ht fast rt pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 7900HT Fast RT-PCR system is a real-time PCR instrument designed for high-throughput gene expression analysis. It can perform fast, sensitive, and accurate quantification of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Revertaid h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Revertaid premium reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Qbase 2, by CellCarta (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Statistica 7, by StatSoft (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Primerquest, by Integrated DNA Technologies (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Gata4, by Santa Cruz Biotechnology (1 mentions)
Quantitect transcription kit, by Qiagen (1 mentions)
Alexa flour fluorescent secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Nestin, by Santa Cruz Biotechnology (1 mentions)
5 protocols using abi 7900ht fast rt pcr system
1
Quantitative Gene Expression Analysis
2
Gene Expression Analysis of PCD Regulation
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The expression of marker genes involved in PCD regulation was measured with real time quantitative PCR (qPCR). Three biological repeats were used for gene expression analysis with qPCR. RNA was treated with DNAseI and reverse transcription was performed using 2 μg of RNA with the RevertAid Premium Reverse Transcriptase (RT) and Ribolock Rnase inhibitor according to manufacturer’s instructions (Thermo Fisher Scientific). After reverse transcription the reaction was diluted to the final volume of 100 μl. 1 μl was used for PCR with EvaGreen ROX (Solis Biodyne). The cycle conditions in the ABI 7900HT Fast RT PCR System (Applied Biosystems) were: 95°C 10 min, 40 cycles with 95°C 15 s, 60°C 30 s, 72°C 30 s and ending with melting curve analysis. Normalization of the data was performed in qBase 2.0 (Biogazelle), with three reference genes TIP41, YLS8 and SAND. Primer amplification efficiencies were determined in qBase from a cDNA dilution series. Primer sequences and amplification efficiencies can be found in S1 Table . Data normality was tested and subsequently 2-base logarithmed for statistical analyses. Factorial ANOVA posthoc analyses Fisher LSD was used to evaluate significant differences between mutant and leaf age, and One-Way ANOVA to changes in gene expression with leaf age (Statistica 7.1, Stat Soft Inc).
3
Quantitative Real-Time PCR Protocol
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The SuperScript II reverse transcriptase kit (Invitrogen) was used to synthesize the first strand of cDNA from 1 μg of DNase and RNase-treated total RNA in a volume of 20 μl. Primers were designed to amplify 100–200 bp fragments, using PrimerQuest (Integrated DNA Technologies) software. The reaction mix for real-time PCR contained 0.4 mM of each primer, 10 μl of SYBR green PCR Master Mix (AppliedBiosystems), 5 μl of a 1:5 dilution of the cDNA product, and DEPC water to a final volume of 20 μl. Cycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The reactions were carried out in fast 96-well reaction plates on the ABI 7900HT fast RT-PCR system (Applied Biosystems). Fungal transcript levels were normalized by using the fungal actin gene as an internal standard, and relative expression was calculated using the Pfaffl method [126 (link)]. Maize genes were normalized against the maize actin gene, and expression was calculated relative to mock-inoculated plants.
4
Validating Bioinformatics Analyses by qPCR
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To verify the reliability of the results of bioinformatics analysis, qPCR experiment of several DEMs was performed. The blood samples from fracture patients with type‐2 diabetes, fracture patients without type‐2 diabetes, and healthy subjects were collected. Subsequently, blood was clotted in an upright position for 40 min, and then centrifuged at 2000 × g for 15 min. Serum supernatant was obtained for further study. Total RNA from the serum was extracted using RNAiso Plus reagent (9109, TaKaRa, Dalian, China) according to the instructions of the manufacturer. The quality and concentration of the RNA were detected using a NanoDrop 1,000 Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Then, the RNA was reverse transcribed into complementary DNA (cDNA) using PrimeScript™RT Master Mix kit (RR036A, TaKaRa, Dalian, China), and qPCR was performed by utilizing a Power SYBR Green PCR Master Mix (A25742, Thermo Scientific, MA, USA) on an ABI 7900HT Fast RT‐PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The process of qPCR amplification is as follow: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. Relative gene expression was calculated by the 2−ΔΔCt method, and U6 small‐nuclear RNA (U6 snRNA) was used as a reference gene to normalize the expression of miRNA. The primer sequences are listed in Table S2 .
5
Comprehensive RNA Extraction and Gene Expression Analysis
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Total RNA was extracted using Trizol (Invitrogen). First-strand cDNA was generated using a Quanti-Tect Transcription Kit (Qiagen). The quantitative real-time PCR (RT-PCR) mixtures were prepared using SYBR Green PCR Master Mix and run on an ABI7900HT Fast RT-PCR System (Applied Biosystems, Carlsbad, CA, http://www.lifetechnologies.com/applied-biosystems ). RT-PCR was carried out using relative expression levels of pertinent genes that were normalized against GAPDH. Immunostaining was performed according to a standard protocol, using primary antibodies including Nestin (1:100, Santa Cruz), Tuj1 (1:500, Covance, Princeton, NJ, www.covance.com ), Myosin (1:100, Developmental Hybridoma Studies Bank) and Gata4 (1:100, Santa Cruz). Alexa Flour fluorescent secondary antibodies (Invitrogen) were used at a dilution of 1:2,000 and nuclei were stained with DAPI (1:5,000).
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