DNA concentration of all samples (biological material and synthesized gBlocks gene fragments) was standardized by qPCR quantification using the lasiocampid general primers (Table 1 ). Standardization of DNA quantities before running the TaqMan assays allowed us to confirm that DNA was present at a high enough concentration to ensure discrimination of all closely related species in the assays. Using the LRE method of Rutledge (2011) , DNA quantity was standardized to ~5000 COI copies, which corresponds to a Ct value of 20. Lasiocampid general primers were designed in a conserved region of the 5’ end of the COI gene (Supp File S1 [online only ]) and generated a 139 bp amplicon. qPCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR machine (Thermofisher, Waltham MA). 96-well MicropAmp Fast Optical real-time PCR plates were used with MicroAmp Optical Adhesive Film (Thermofisher, Waltham, MA). The 10 µl amplification reactions contained 2 µl DNA and 500 nM of primers. PCR conditions were as follows: a 15 min initial denaturation step at 95°C for enzyme activation, followed by 45 cycles of 95°C for 15 s, 50°C for 30 s, and 65°C for 90 s. PCR was performed using the Qiagen Quantitect SYBR Green PCR Kit (Qiagen, Venlo, Netherlands).
Applied biosystems 7500 fast real time pcr machine
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems 7500 Fast Real-Time PCR System is a laboratory instrument used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It enables accurate and rapid detection and quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna rt kit, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Flashgel, by Lonza (2 mentions)
Accuprime pfx supermix, by Thermo Fisher Scientific (2 mentions)
Microamp optical adhesive film, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Quick rna microprep kit, by Zymo Research (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Sensimix sybr low rox kit, by Meridian Bioscience (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Sangon (1 mentions)
Prism 8, by GraphPad (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Magmax 96 total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Dnazol genomic dna isolation reagent, by Molecular Research Center (1 mentions)
Maxima sybr green qpcr master mix kit, by Thermo Fisher Scientific (1 mentions)
13 protocols using applied biosystems 7500 fast real time pcr machine
1
Standardized DNA Quantification for Assay Optimization
2
14-3-3ζ Regulation of U87 Glioblastoma
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U87 glioblastoma
cells were cultivated in DMEM (+10% FCS) at 37 °C at 5% CO2. Cells were plated for 24 h, and medium was changed (DMEM
+ 1% FCS). After another 24 h, cells were treated with 200 nM 14–3–3ζ,
the corresponding peptides in DMEM + 1% FCS and 0.5% DMSO. Untreated
and 14–3–3ζ-treated controls were cultivated under
the same conditions with 0.5% DMSO. After 24 h of incubation, total
RNA was isolated (Quick-RNA MicroPrep Kit, Zymo Research) and reverse
transcribed into cDNA (Quanti Tect Reverse Transcription Kit, Qiagen).
Next, cDNA was used for quantitative real time PCR (SensiMix SYBR
Low-ROX Kit, Bioline) in the Applied Biosystems 7500 Fast Real Time
PCR machine (Thermo Fisher Scientific). For relative quantitation,
2−ΔΔCT method was used with the reference
gene GAPDH.
cells were cultivated in DMEM (+10% FCS) at 37 °C at 5% CO2. Cells were plated for 24 h, and medium was changed (DMEM
+ 1% FCS). After another 24 h, cells were treated with 200 nM 14–3–3ζ,
the corresponding peptides in DMEM + 1% FCS and 0.5% DMSO. Untreated
and 14–3–3ζ-treated controls were cultivated under
the same conditions with 0.5% DMSO. After 24 h of incubation, total
RNA was isolated (Quick-RNA MicroPrep Kit, Zymo Research) and reverse
transcribed into cDNA (Quanti Tect Reverse Transcription Kit, Qiagen).
Next, cDNA was used for quantitative real time PCR (SensiMix SYBR
Low-ROX Kit, Bioline) in the Applied Biosystems 7500 Fast Real Time
PCR machine (Thermo Fisher Scientific). For relative quantitation,
2−ΔΔCT method was used with the reference
gene GAPDH.
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) was employed to determine the purity and concentration of the RNA. cDNA was generated from 1 g of total RNA by using a High-capacity cDNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. qPCR was performed on an Applied Biosystems 7500 Fast real-time PCR machine (Thermo Fisher Scientific, Inc.) using SYBR Green Master Mix (Sangon Biotech Co., Ltd.) according to the manufacturer's instructions. For the qPCR, the thermocycling conditions were as follows: 10 min of initial denaturation at 95°C and 40 cycles of denaturation at 95°C for 15 sec and 1 min of annealing and extension at 60°C. The primer sequences used were as follows: GNL2 forward, 5′-ATCCAAATGTTGGCAAGAGC-3′ and reverse, 5′-ACACCTGGACAGTCAATCAGG-3′; GAPDH forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGAG-3′. The relative gene expression was calculated using the 2−ΔΔCq method (13 (link)), with GAPDH as the internal control.
4
Validating RNA-seq Gene Expression by qPCR
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To validate the differentially expressed genes obtained via RNA-seq analysis within the original RNA samples, RNA was converted to cDNA by using the ProtoScript II First Strand cDNA Synthesis kit (New England Biolabs, MA, United States). qPCR was performed in triplicates by using GoTaq qPCR Master Mix (Promega, WI, United States) in an Applied Biosystems 7,500 Fast Real-Time PCR machine (Thermo Fisher Scientific, MA, United States). Expression values of each sample were normalized to Danio rerio ribosomal protein L13a (rpl13a). The efficiency of each primer pair was assessed by using the standard curve assay according to the relevant program of the machine. Standard curve with CT values were generated by using the ABI software and a correlation coefficient (R2) was calculated for each primer pair. Primer pairs with the R2 values equal to or greater than 0.99 and an efficiency falling in the acceptable range (90–110%) were used in the qPCR reactions. Data were analyzed with the GraphPad Prism 8 software (Graphpad Software Inc., CA, United States). The values are indicated as mean ± SEM (Standard Error of Mean) of triplicates. Primer sequences for the tested zebrafish genes are provided in Supplementary Table S1 .
5
Transcriptional Analysis of Pluripotent and Neural Stem Cells
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Total RNA was isolated using MagMAX™ 96 Total RNA isolation Kit (Thermo Fisher Scientific, cat. no. AM1830). High‐throughput MagMAX™ Express 96 was employed to extract RNA from the cell lysate.
EXPRESS One Step Superscript™ RT‐qPCR Kit (Thermo Fisher Scientific, cat. no. 11781 200) and TaqMan™ probes were recruited to perform cDNA synthesis and RT‐qPCR in one step. Applied Biosystems 7500 Fast Real‐Time PCR machine (Thermo Fisher Scientific) was used to perform qRT‐PCR. TaqMan™ probes (Life Technologies) for POU5F1 (Hs00999634), NANOG (Hs04260366), PAX6 (Hs01088114), NESTIN (Hs04187831), and LIN28A (Hs00702808) were used. The average CT values of three technical replicates were normalized to the geometric mean of endogenous control gene, Actin Beta (ACTB: Hs01060665). The expression of the iPSC markers was assessed with fold change by using the comparative ΔΔCt method by normalizing the gene level from ESC1. The expression of the NSC markers was assessed with fold change by using the comparative ΔΔCt method by normalizing NSCs to iPSCs.
EXPRESS One Step Superscript™ RT‐qPCR Kit (Thermo Fisher Scientific, cat. no. 11781 200) and TaqMan™ probes were recruited to perform cDNA synthesis and RT‐qPCR in one step. Applied Biosystems 7500 Fast Real‐Time PCR machine (Thermo Fisher Scientific) was used to perform qRT‐PCR. TaqMan™ probes (Life Technologies) for POU5F1 (Hs00999634), NANOG (Hs04260366), PAX6 (Hs01088114), NESTIN (Hs04187831), and LIN28A (Hs00702808) were used. The average CT values of three technical replicates were normalized to the geometric mean of endogenous control gene, Actin Beta (ACTB: Hs01060665). The expression of the iPSC markers was assessed with fold change by using the comparative ΔΔCt method by normalizing the gene level from ESC1. The expression of the NSC markers was assessed with fold change by using the comparative ΔΔCt method by normalizing NSCs to iPSCs.
6
APOE Genotyping using Taqman Assay
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The method for detecting the APOE polymorphism has already been described in previous work [7 (link),16 (link)]. The genotype was determined using Taqman assay technology. For this purpose, DNA was extracted from a peripheral blood sample collected in an EDTA tube using standard protocols. The C____904973_10 and C___3084793_20 assays were used to analyze the rs7412 and rs429358 SNPs, which conform the haplotype that determines the APOE isoforms. Allelic detection was performed using an Applied Biosystems 7500 Fast Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA), using the appropriate assay quality controls.
7
Genotyping APOE Polymorphisms via TaqMan
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APOE polymorphism (haplotype) was determined by an analysis of the genotype of the two polymorphisms (SNPs) that determine it: rs7412 and rs429358. The DNA was extracted from peripheral leukocytes using DNAzol® Genomic DNA Isolation Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA), following the protocol of the manufacturer. ApoE alleles were determined using TaqMan assay technology on an Applied Biosystems 7500 Fast Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). A genotyping call rate of over 95% per plate, sample controls for each genotype, and negative sample controls were included in each assay. Three well-differentiated genotyping clusters for each SNP were required to validate the results. Intra- and interplate duplicates of several DNA samples were also included.
8
Quantitative RT-PCR for Gene Expression
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Total RNA was extracted from frozen tissue by using Gene JET RNA Purification Kit (catalog no. #K0731, Thermo Scientific Inc., USA) according to the manufacturer’s instructions. After quantitation using NanoDrop spectrophotometer, RNA (500 ng) was used for reverse transcription to complementary DNA (cDNA) with the High-Capacity cDNA Reverse Transcription Kit (catalog no. #K1622, Thermo Scientific, USA). cDNA was then amplified with Maxima SYBR Green qPCR Master Mix Kit (catalog no. #K0251, Thermo Scientific, USA) and used as a template for the genes in Table 1 . A two-step reaction protocol was performed: an initial denaturation cycle of 95 °C for 10 min, followed by 40 amplification cycles of 95 °C for 15 s and 60 °C for 1 min using the Applied Biosystems 7500 Fast Real-Time PCR Machine (Applied Biosystems, Germany). Calculations were performed using the 2−ΔΔCT method with GAPDH as reference gene (Table 1 ).
Primer sequences
| Gene | 5′-3′ primer sequence |
|---|---|
| NEP | Forward: CTG CTG AGG GGT CAC GAT T Reversed: GAG TGC GAT CAT TTC ACA GC |
| IL-6 | Forward: AAA GAG GCA CTG GCA GAA AA Reversed: TTT CAC CAG GCA AGT CTC CT |
| IL-10 | Forward: TTG CAA AAC CAA ACC ACA AGA CA Reversed: TCT CGA AGC ATG TTA GGC AGG |
| TGF-β | Forward: TCC TGG CGA TAC CTC AGC AA Reversed: CTC AAT TTC CCC TCC ACG GC |
| GAPDH | Forward: ACA GTC AGC CGC ATC TTC TT Reversed: GAC AAG CTT CCC GTT CTC AG |
9
Antioxidant Gene Expression in Hippocampal Samples
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CD11b mRNA expression, as well as expression of HO-1, NQO1 and SOD, were assessed in hippocampal samples prepared from DMF- or vehicle-treated mice by RT-PCR. RNA was isolated using the Nucleospin® RNAII KIT (Macherey-Nagel, Duren, Germany) and cDNA was prepared using the High-Capacity cDNA RT kit according to the manufacturer’s instructions (Applied Biosystems, Warrington, UK). Real-time PCR was performed with predesigned Taqman gene expression assays (CD11b: Mm00434455_ml; HO-1; Mm00516005, NQO1; Mm01253561, and SOD; Mm01344233; Applied Biosystems, Warrington, UK) using an Applied Biosystems 7500 Fast Real-Time PCR machine (Applied Biosystems, Warrington, UK). Samples were assayed as previously described (Costello et al., 2016) with β-actin (Mm00407939_s1) as the endogenous control to normalize gene expression data. Gene expression was calculated relative to the endogenous control samples and to the control sample.
10
Quantification of CD274 Expression
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RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Reverse transcription reactions were conducted using Advantage RT‐for‐PCR kit (Takara Bio, Tokyo, Japan). The cDNA was amplified using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with the Applied Biosystems 7500 Fast real‐time PCR machine (Applied Biosystems) and predeveloped TaqMan assay primers and probes (human CD274, Hs00234244_m1; mouse CD274, Mm00452054_m1; all from Applied Biosystems). Data were normalized to 18s gene expression.
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