Total RNA was extracted from control or BRCA1 siRNA transiently transfected breast cancer cells (MCF7 or MDA-MB-231) using Quick-RNA Miniprep Kit (Zymo Research). cDNAs were generated by Invitrogen™ SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR kit following supplier’s instruction (Fisher Scientific). For each qPCR reaction, 2 μL of cDNA from each sample was used with SYBR Green Supermix. Each reaction was prepared in triplicates in each experiment. Primer pairs targeted EIF1AK3 (PERK), ERN1 (IRE1), BRCA1, BARD1 and GAPDH were used in qPCR reactions. All reactions were run in 96-well plate and analyzed by Applied Biosystem 7500 Real-Time PCR System (ThermoFisher Scientific). Each analysis was performed at least 3 times.
Applied biosystem 7500 real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystem 7500 Real-Time PCR System is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It performs sensitive and reliable detection and quantification of nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Sybr green, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Maxima sybr green rox master mix, by Thermo Fisher Scientific (3 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (2 mentions)
Quantifiler human dna quantification kit, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix kit, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Rt2 first strand kit, by Qiagen (2 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Easyscript reverse transcriptase kit, by Transgene (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis supermix for qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Reverse transcription kit, by Abcam (1 mentions)
Sybr premix ex taq master mix, by Takara Bio (1 mentions)
40 protocols using applied biosystem 7500 real time pcr system
1
Quantifying BRCA1 Pathway in Breast Cancer
2
Quantification of Gene Expression
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Total RNA was isolated from cells and tissues using TRIzol reagent, after which mRNA (1 μg) was reverse transcribed to cDNA using PrimeScript RT Master Mix (Takara Bio, Tokyo, Japan). Transcript expression was assessed by quantitative PCR using an Applied Biosystem 7500 Real-Time PCR System (Thermo Fisher Scientific, USA). Target (rat TGF-β1, PLOD2, MAP2, NSE, GFAP, and GAP43) cDNA amplification was measured using SYBR Premix Ex Taq II (Takara Bio, Japan). Fold-change expression for each target mRNA was calculated with the CT (2-ΔΔCT) method, using β-actin levels for normalization. PCR assays were performed at least 3 times. Primer sequences are listed in Supplementary Table 1 .
3
Quantitative PCR Analysis of RNA Expression
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Total RNA from tissue samples and cultured cells was isolated using TRIzol reagent (Thermo, Waltham, MA, USA) following the protocol. The first strand of cDNA was synthesized with total RNA as a template using a reverse transcription kit (Abcam, Cambridge, UK), and amplification of cDNA was performed by SYBR Premix Ex Taq Master Mix (TaKaRa, Shiga, Japan). qPCR was conducted on an Applied biosystem 7500 real-time PCR system (Thermo), and the expression of DLX6-AS1, Rab10 mRNA and miR-141-3p was calculated according to the comparative threshold cycle value (2−ΔΔCt) method, as compared with GAPDH or U6 small nuclear RNA (U6, for miRNA). PCR primers were as follows: DLX6-AS1: forward, 5′-AGTTTCTCTCTAGATTGCCTT-3′; reverse, 5′-ATTGACATGTTAGTGCCCTT-3′;25 (link) miR-141-3p: forward, 5′-GGGCATCTTCCAGTACAGT-3′; reverse, 5′-CAGTGCGTGTCGTGGAGT-3′;26 (link) Rab10: forward, 5′-CAAGGGAGCATGGTATTAGGTTT-3′; reverse, 5′-CTAACGTGAGGAACGCCTTTT-3′;27 (link) GAPDH: forward, 5′-AGAGGCAGGGATGATGTTCTG-3′; reverse, 5′-GACTCATGACCACAGTCCATGC-3′; U6: forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′-AACGCTTCACGAATTTGCGT-3′. All operations were carried out at least 3 times.
4
Quantitative RT-PCR Analysis of Gene Expression
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Analysis of qRT-PCR was performed using a SYBR Green PCR Mix (Simgen, Hangzhou, China). CsGAPDH was used as the internal reference gene. Real-time qPCR was performed on an Applied Biosystem 7500 Real-Time PCR system (Thermo Fisher Scientific, USA) using the following program: 95 °C for 30 s ; 40 cycles of 95 °C for 5 s each, annealing at 60 °C for 34 s; 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The 2−ΔΔCt method52 (link) was employed to calculate the relative expression. The specific primer sequences are shown in Table S1 . All measured expression levels were determined in six replicates and the results were presented as mean ± SD.
5
SARS-CoV-2 Diagnostic Platforms Evaluation
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Nasopharyngeal swabs transported in viral transport medium were processed at the molecular virology laboratory at KFSHRC. Four platforms were used for the routine diagnosis of SARS-CoV-2. The first platform used the Abbott m2000 system (Abbott, IL) for high-throughput RNA extraction, and elutions were tested using the RealStar SARS-CoV-2 RT-PCR kit (Altona Diagnostics, Germany) and amplified on the Rotor-Gene Q real-time PCR cycler (Qiagen, Germany). The second platform used the Applied Biosystems MagMAX viral RNA kit on the KingFisher instrument (Thermo Fisher Scientific) for high-throughput RNA extraction, and elutions were tested using the DiaPlexQ novel coronavirus detection kit (2019-nCoV) (SolGent, South Korea) and amplified on Applied Biosystem 7500 real-time PCR system (Thermo Fisher Scientific). The third platform is the sample-to-result Abbott m2000 RNA extraction system combined with the Abbott RealTime SARS-CoV-2 assay, and the fourth platform is the point-of-care GeneXpert using the Xpert Xpress SARS-CoV-2 test (Cephid, CA). The samples were tested according to the manufacturer’s protocols.
6
Genomic DNA Extraction and Quantification
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Peripheral blood samples were collected from 206 unrelated Han Chinese individuals (104 females and 102 males) residing in Guizhou province, southwest China. We used PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) to extract and isolate human genomic DNA, and used an Applied Biosystem 7500 Real-time PCR System (Thermo Fisher Scientific) and Quantifiler Human DNA Quantification Kit (Thermo Fisher Scientific) to measure the DNA concentration on the manufacturer’s protocol. Finally, we diluted the DNA to 2.0 ng/μL and stored at -20°C until amplification.
7
Cardiac Gene Expression Analysis
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RNA was isolated from heart samples using the NucleoSpin TriPrep kit with on-column DNase digestion (Macherey-Nagel, Dueren, Germany). Extracted RNAs were quantified and checked by 260/280 nm measurement using the Synergy H1 spectrophotometer with Take 3 microvolume plate (BioTek; Agilent Technologies, Santa Clara, CA, USA). Reverse-transcription PCR (RT-PCR) was performed using QuantiTect RT kit (Qiagen, Hilden, Germany), and quantitative PCR (qPCR) was performed using BlitzAmp Hotstart qPCR mix (MiRXES, Singapore) with an Applied Biosystem 7500 Real-Time PCR system (Thermo Fisher Scientific, MA, USA). Primers sequences are listed in Appendix A (Table A1 ), with gene expression normalized to β-actin.
8
SARS-CoV-2 Detection via RT-PCR
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Oropharyngeal swab samples were taken and extracted, per manufacturer instructions, on the KingFisher™ Flex Purification System (ThermoFisher Scientific, USA) using the MagMAX Viral/Pathogen Nucleic Acid Isolation kit (ThermoFisher Scientific, USA). The RT-PCR testing was performed on Applied biosystem 7500 Real-Time PCR system (Thermo Fisher Scientific, USA) using Genesig® COVID-19 2G RealTime CE-IVD PCR Assay (Primerdesign Ltd, UK), which targets the ORF1ab and S gene of SARS-CoV-2.
9
Quantitative Analysis of RNA Transcripts
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Total RNA from tissue samples and cultured cells was isolated using TRIzol reagent (Thermo, Waltham, MA, USA) following the manufacturer's protocol. The first strands of cDNA were synthesized dependent on total RNA using reverse transcription kits (Abcam, Cambridge, UK), and amplification of cDNA was performed using SYBR Premix Ex Taq Master Mix (Invitrogen). Real-time quantitative PCR (qPCR) was conducted on an Applied Biosystem 7500 Real-Time PCR System (Thermo), and the expression of selected mRNAs and miR-342-3p were calculated according to the comparative threshold cycle value (2−ΔΔCt) method, compared with β-actin (for mRNA) or U6 small nuclear RNA (U6, for miRNA). PCR primers for LINC00313, miR-342-3p, U6, and β-actin were purchased from Invitrogen. All operations were conducted at least three times.
10
Quantitative Analysis of MSX1 Expression
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Total RNA was extracted from tissue and infected cells with TRIzol reagent in accordance with the manufacturer’s specifications. RT-PCR was performed as described previously23 (link) using GAPDH as an internal control. The four primer sequences used for polymerase chain reaction (PCR) amplification in this study are given in Table 1 . RT-PCR was carried out with 23 cycles for GAPDH and 32 cycles for gene MSX1 with Go-Taq DNA polymerase. The PCR program began with an initial denaturation at 95°C for 2 min, followed by amplification reaction cycles (95°C for 30 s, 55°C for 30 s, and 72°C for 30 s) with a final extension at 72°C for 3 min. Quantitative PCR was performed using a SYBR® Green PCR Master Mix kit (Thermo Fisher Scientific) and an Applied Biosystem 7500 Real-time PCR System (Thermo Fisher Scientific). β-actin served as a control. The relative expression of MSX1 was evaluated using the method. All assays were performed three times independently. The primer sequences are shown in Table 1 .
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