Total RNA was extracted from cells using TRIzol Reagent (Invitrogen) and cDNA was prepared from total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative PCR (qPCR) was performed for mRNA levels using an Applied Biosystems 7900HT Fast Real-Time PCR System (Carlsbad, CA) and QuantiTect Primers Assays (Qiagen Inc., Valencia, CA) for AR, NQO1 and GAPDH mRNA. Additional forward and reverse primers used for qPCR are listed in Table in S1 Table .
7900ht fast real time pcr system
Manufactured by Qiagen
Sourced in Germany
The 7900HT Fast Real-Time PCR System is a high-performance instrument designed for real-time PCR applications. It features a fast thermal cycler and sensitive optical detection system to enable rapid and accurate gene expression analysis and quantification of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Quantifast sybr green pcr kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Direct zol rna microprep kit, by Zymo Research (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sars cov cdc ruo primers and probes, by Integrated DNA Technologies (2 mentions)
Quantitect virus kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Quantitect primers assay, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Superscript 4 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Miscript pcr system, by Qiagen (1 mentions)
Fluorchem sp imaging system, by Bio-Techne (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
S1000 thermal cycler, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
8 protocols using 7900ht fast real time pcr system
1
Quantitative PCR Analysis of Gene Expression
2
SARS-CoV-2 RNA Detection by RT-qPCR
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RNA was collected in 100 μl of Trizol (Thermo Fisher Scientific, Waltham, MA) per insert and incubated for 15 mins at room temperature. Cell isolates were gently mixed by pipetting up and down. An additional 900 μl of Trizol was added and cell isolates were collected and stored at −80°C until required. Cellular RNA was isolated by either phenol/chloroform extraction or using the Direct-zol RNA Microprep kit (Zymo Research, Irvine, CA). RT-qPCR was performed in 384 well plates on an Applied Biosystems 7900HT Fast Real-Time PCR system using the QuantiTect Virus Kit (Qiagen, Redwood City, CA) and SARS-CoV-CDC RUO primers and probes (Integrated DNA Technologies (IDT), Coralville, IA). Briefly, each 5 μl reaction contained 1 μl 5x QuantiTect Virus Master Mix, 500 nM forward primer, 500 nM Reverse Primer, 125 nM Probe, 10 ng DNA, 0.05 μl QuantiTect Virus RT Mix, and DNAse/RNAse-free water up to a final volume of 5 μl. Calibration curves for RNAseP primers/probe was performed with 10-fold dilutions of RNA from uninfected Calu3 cells (ATCC, Manassas, VA) from 100 ng to 0.01 ng per reaction. Calibration curves for N1 primers were performed on 5 ng of RNA from uninfected Calu3 cells per reaction spiked with 10-fold dilutions from 50 ng to 0.005 ng of RNA from Calu3 cells collected 48 hours post infection. Relative gene expression was calculated using the Pfaffl method [29 (link)].
3
Gene Expression Analysis by RT-PCR
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Total RNA was extracted with TRIzol reagent (Invitrogen Corp., Carlsbad, CA), and cDNAs were synthesized by using the SuperScript® IV First-Strand Synthesis System for RT-PCR (Invitrogen Corp.). A pair of oligonucleotides (forward: 5′-AGA AGG CTG GGG CTC ATT TG -3′ and reverse: 5′-AGG GGC CAT CCA CAG TCT TC -3′) were adopted to amplify human GAPDH cDNA as a loading control. The human RAC1 specific PCR primers (forward: 5′- ATC AAG TGT GTG GTG GTG GG -3′; reverse: 5′-CCA GCT GTA TCC CAT AAG CCC A -3′) The human Dicer specific PCR primers (forward: 5′-GAG TGT TTG AGG GAT AG -3′; reverse: 5′-CTG AGG TAT GGG TTT GG-3′). The PCR products were then separated on 2% agarose gels and stained with ethidium bromide. The images were visualized and scanned by using the UV lights with a FluorChem SP imaging system (Alpha Innotech, San Leandro, CA) as previously described [32 ]. The Quantitative RT-PCR analysis was conducted for miRNA assay. Total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA), and analysis of miRNAs expression was performed by the miScript PCR system (Qiagen) and the 7900HT Fast Real-time PCR system. The ΔΔCT value was used to calculate the relative expression of miRNAs, using U6 as endogenous control.
4
Quantification of Osteogenic Gene Expression
5
Quantitative Real-Time PCR Analysis
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Cells were incubated at a density of 1 × 105 cells per well. Following overnight serum starvation, cells were treated with the indicated concentrations of LPA for indicated time periods. Total RNA was extracted using the RNeasy Mini kit (QIAGEN, Hilden, Germany) according to manufacturer’s protocol and quantified using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). 1 µg of RNA was reverse-transcribed by using SuperScript® III reverse transcription kit (Invitrogen, Waltham, MA, USA). Quantitative real-time PCR (qPCR) was performed on Applied Biosystems 7900HT Fast Real-Time PCR System using QuantifastTM SYBR® Green PCR kit (QIAGEN, Hilden, Germany. Relative gene expression levels were normalized to hypoxanthineguanine phosphoribosyltransferase (HPRT) and calculated using ΔΔCT method [85 (link)]. Primer sequences are listed in Table 1 .
6
Brain Total RNA Extraction and qPCR Analysis
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Total RNA from the brain was extracted using the RNeasy lipid tissue mini kit (QIAGEN, Hilden, Germany) according to manufacturer’s protocol and quantified using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse-transcribed by using SuperScript® III reverse transcription kit (Invitrogen, Waltham, MA, USA). Quantitative real-time PCR (qPCR) was performed on Applied Biosystems 7900HT fast real-time PCR system using QuantifastTM SYBR® Green PCR kit (QIAGEN, Hilden, Germany). Relative gene expression levels were normalized to hypoxanthineguanine phosphoribosyltransferase (HPRT) and calculated using ΔΔCT method [63 (link)]. Primer sequences are listed in Table 2 .
7
Quantitative Gene Expression Analysis
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After transfection with the indicated siRNAs, total RNA was extracted and reverse transcribed. Quantitative PCRs (qPCRs) were performed using the Applied Biosystems 7900HT Fast Real Time PCR System, the QuantiFast SYBR Green PCR Kit, and Quantitect Primer Assays (Qiagen). Relative changes in gene expression were normalized to hypoxanthine phosphribosyltransferase 1 (HPRT1).
8
SARS-CoV-2 Detection by RT-qPCR
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RNA was collected in 100 µl of Trizol (Thermo Fisher Scientific) per insert and incubated for 15 mins at room temperature. Cell isolates were gently mixed by pipetting up and down. An additional 900 µl of Trizol was added and cell isolates were collected and stored at -80°C until required. Cellular RNA was isolated by either phenol/chloroform extraction or using the Direct-zol RNA Microprep kit (Zymo Research, Irvine, CA). RT-qPCR was performed in 384 well plates on an Applied Biosystems 7900HT Fast Real-Time PCR system using the QuantiTect Virus Kit (Qiagen, Redwood City, CA) and SARS-CoV-CDC RUO primers and probes (Integrated DNA Technologies (IDT), Coralville, IA). Briefly, each 5 µl reaction contained 1 µl 5x QuantiTect Virus Master Mix, 500 nM forward primer, 500 nM Reverse Primer, 125 nM Probe, 10 ng DNA, 0.05 µl QuantiTect Virus RT Mix, and DNAse/RNAse-free water up to a final volume of 5 µl. Calibration curves for RNAseP primers/probe was performed with 10-fold dilutions of RNA from uninfected Calu3 cells (ATCC, Manassas, VA) from 100 ng to 0.01 ng per reaction. Calibration curves for N1 primers were performed on 5 ng of RNA from uninfected Calu3 cells per reaction spiked with 10-fold dilutions from 50 ng to 0.005 ng of RNA from Calu3 cells collected 48 hours post infection. Relative gene expression was calculated using the Pfaffl method (28 (link)).
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