Total RNA was extracted from the frozen brain stem using the High Pure RNA Tissue Kit (Roche Molecular Biochemicals, GmbH, Mannheim, Germany). Approximately 30 mg of tissue was homogenized using a homogenization pestle, and RNA was extracted according to the manufacturer’s recommendations. Then, 50 μL of RNA was eluted and stored at −30°C in a low-temperature freezer until further use. For real-time RT-PCR, the LN34 assay was performed using AgPath-ID One-step RT-PCR Reagents (Applied Biosystems, Foster City, CA, USA) [41 (link)–43 ]. The master mix consisted of the following: 6.5 μL of ddH2O, 12.5 μL of 2× RT buffer, 1 μL of 25× RT-PCR Enzyme Mix, 1 μL of either LN34 or beta-actin primer sets (10 μM), 1 μL of either LN34 or beta-actin probe (5 μM), and 2 μL of RNA template [41 (link)–43 ]. The sealed plate was placed into an ABI Step One Plus Real-Time PCR (Applied Biosystems Foster City, CA, USA), and the following conditions were set: reverse transcription at 50°C for 30 min, denaturation at 95°C for 10 min, and amplification of 45 cycles at 94°C for 15 s and 56°C for 30 s using ABI7500-standard mode. To estimate viral load, the Cq values were divided into >25 (low copy numbers), 15–25 (high copy numbers), and <15 (very high copy numbers).
Abi steponeplus real time pcr
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI StepOnePlus Real-Time PCR System is a compact and versatile instrument used for real-time polymerase chain reaction (PCR) analysis. It is designed for high-throughput gene expression, genotyping, and copy number variation studies. The system utilizes fluorescence detection technology to monitor the amplification of DNA samples in real-time, enabling accurate and precise quantification of target sequences.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Taqman fast advance master mix, by Thermo Fisher Scientific (2 mentions)
Rnaqueous micro total rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Kapa sybr fast master mix, by Roche (2 mentions)
Omniscript reverse transcriptase, by Qiagen (2 mentions)
Agpath id one step rt pcr reagent, by Thermo Fisher Scientific (1 mentions)
High pure rna tissue kit, by Roche (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 3 rt platinum taq mix, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Rna extraction kit, by Tiangen Biotech (1 mentions)
Reverse transcription kit, by Tiangen Biotech (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent solution, by Thermo Fisher Scientific (1 mentions)
19 protocols using abi steponeplus real time pcr
1
RNA Extraction and RT-PCR for Viral Load Quantification
2
AT1R mRNA Quantification in Hippocampus and Amygdala
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AT1R mRNA was measured by real-time PCR. Total RNA from the CA1 & CA3 regions of the Hc and the BLA were extracted using an RNAqueous™-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA). First-strand cDNA was made from 500 ng of total RNA using a iScript cDNA synthesis kit (BioRad, Hercules, CA) with Moloney Murine Leukemia Virus (MMLV) RNase H + reverse transcriptase, oligo(dT) and random hexamers. RNA concentrations were measured on a NanoDrop 2000 (Thermo Scientific). Quantitation of specific mRNAs was performed by real-time PCR using the ABI StepOnePlus real-time PCR (Applied Biosystems Inc., Foster City, CA). The real-time PCR mixture consisted of RNase-free water, TaqMan Fast Advance Master Mix (Thermo Fisher Scientific), and the following specific primers (300 nM) and probe (10 µM): forward primer: rAT1R-CR-330F 5′-CAACCTCTACGCCAGTGTGTTC-3′; reverse primer: rAT1R-CR-470R:5′-CCAGCCATCAGCCAGATGA-3′; and probe: rAT1aR-CR-382T: Fam-CTGGCCATCGTCCACCCAATGAAGT-Tamra and cDNA samples. PCRs without reverse transcription and no template were included to control for genomic DNA contamination. The standard curve with rAT1aR plasmid DNA was used to determine the DNA copy number in the Hc and BLA samples. The data were expressed in DNA copy numbers × 106 and normalized to µg of total RNA in each sample.
3
qPCR Amplification Protocol for I. nubecula
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qPCR amplifications were performed on an ABI StepOnePlus™ Real-Time PCR (Applied Biosystems) in final volumes of 25 µL, using 12.5 µL of PrecisionPlus qPCR Master Mix with ROX (Primer Design, UK), 1 µL of each primer (10 µM), 1 µL of probe (2.5 µM), 6.5 µL of ddH2O and 3 µL of extracted DNA. qPCR conditions were as follow: 50 °C for 2 min and 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. For each qPCR with DNA from tissue samples, at least two positive and two negative controls were included. A standard curve was established by analysing a 1:10 dilution series of DNA extracted from I. nubecula (68.2 ng/ µL, Nanodrop 2000 Spectrophotometer, ThermoFisher Scientific) following the MIQE Guidelines33 (link) (Appendix 2 ).
4
Quantitative Gene Expression Analysis
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Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA); 1 µg of RNA was used for first-strand cDNA synthesis, according to the manufacturer's protocol (#639543; Takara Bio, Inc., Shiga, Japan). Using a thermocycler (ABI StepOnePlus Real-Time PCR; Applied Biosystems, Foster City, CA, USA) with the TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA) and the suggested PCR conditions, the resulting cDNA amplification was performed to quantitatively determine the level of gene transcript in the samples. All PCRs were carried out in triplicate using Hs00985639_m1 for IL-6, Hs00174103_m1 for IL-8, Hs00234140_m1 for monocyte chemotactic protein (MCP)-1, Hs00153133_m1 for COX-2, and H299999905_m1 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as primers.
The amount of cytokine mRNA normalized to GAPDH was calculated and expressed as fold changes in the threshold cycle (Ct) value relative to the control group using the 2−∆∆Ct method.15 (link)
The amount of cytokine mRNA normalized to GAPDH was calculated and expressed as fold changes in the threshold cycle (Ct) value relative to the control group using the 2−∆∆Ct method.15 (link)
5
Quantitative Real-Time PCR Assay
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The qRT-PCR assay was performed in a one-step reaction using the ABI StepOne Plus Real-Time PCR (Applied Biosystems), in triplicate (technical replicates) using samples from three independent experiments (biological replicates). Each reaction contained 6 μl of 2X SYBR Green Reaction Mix with Rox, 0.25 μl of SuperScript III RT/Platinum Taq Mix (Invitrogen), 100 nM of each primer, 1 ng μl−1 RNA and sufficient DEPC water for a final volume of 12 μl. The rpoA gene was used as the endogenous control. Data collection was performed using the ABI StepOneTM Real-Time PCR software v.2.1 (Applied Biosystems). Data were normalized to levels of rpoA and analyzed using the comparative critical threshold (CT) method [62 (link)]. Error bars represent the standard deviations (SD) of the CT values.
6
HCMV Viral Load Monitoring by Real-time PCR
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Since HCMV-DNAemia is an independent risk factor for HCMV infection [18 (link)], virological surveillance for virus replication is routinely performed in the first 100 days post-transplant using real-time PCR for monitoring of HCMV-DNA in the blood samples.
Using 200 μl of each patient’s plasma sample, HCMV genomic DNA was extracted using QIAamp DNA mini kit (Qiagen, Germany) according to the manufacturer’s instructions. TaqMan®-MGB probed real-time PCR was applied using HCMV RQ PCR kit (Novin-Gene, Iran) according to the manufacturer’s instructions. The limit of detection (LOD) of the kit was 200 copies /ul.
The thermal cycling program started with 10 min at 95 °C for initial denaturation followed by 45 cycles of denaturation (15 s at 95 °C), annealing (60 s at 58 °C), and extension (30 s at 58 °C). The program was run in the ABI Step One Plus real-time PCR (Applied Biosystems, USA) in triplicate. To quantify HCMV loads, a standard curve was created using standards of the kit that contained 10,000, 1000, 100, and 10 virus DNA copies/ul.
The results were included when the efficiency (E) was more than 90% and R2 was more than 88%.
Using 200 μl of each patient’s plasma sample, HCMV genomic DNA was extracted using QIAamp DNA mini kit (Qiagen, Germany) according to the manufacturer’s instructions. TaqMan®-MGB probed real-time PCR was applied using HCMV RQ PCR kit (Novin-Gene, Iran) according to the manufacturer’s instructions. The limit of detection (LOD) of the kit was 200 copies /ul.
The thermal cycling program started with 10 min at 95 °C for initial denaturation followed by 45 cycles of denaturation (15 s at 95 °C), annealing (60 s at 58 °C), and extension (30 s at 58 °C). The program was run in the ABI Step One Plus real-time PCR (Applied Biosystems, USA) in triplicate. To quantify HCMV loads, a standard curve was created using standards of the kit that contained 10,000, 1000, 100, and 10 virus DNA copies/ul.
The results were included when the efficiency (E) was more than 90% and R2 was more than 88%.
7
Quantitative Analysis of Gene Expression
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LSK cells were isolated by fluorescence cell sorting and were used to extract the cDNA as described previously. Total RNA was isolated by using the mirVana miRNA isolation kit (Invitrogen). cDNA reverse transcription were performed by using the high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative real-time PCR was performed with the commercially available Taqman probe for Thbs1, Lxn, and Gapdh by using the TaqMan Universal PCR Master Mix (Applied Biosystems) in ABI StepOnePlus Real-Time PCR (Applied Biosystems).
8
Quantification of AT1R mRNA in Brain Regions
9
Gene Expression Analysis by qRT-PCR
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Total cellular RNA was extracted using an RNA Extraction kit (Tiangen Biotech Co., Ltd.). First-strand cDNA was synthesized using a Reverse Transcription kit (Tiangen Biotech Co., Ltd.) and subjected to real-time PCR analysis. PCR was performed in triplicate with an ABI Step One Plus Real-Time PCR (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the following conditions: 95°C for 2 min followed by 40 cycles at 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec, with a final extension at 72°C for 10 min as previously described (16 (link),17 (link)). Sequences of the gene-specific primers (sense and antisense, respectively) were: 5′-TCTTCCAGCCTTCCTTCCT-3′ and 5′-AGCACTGTGTTGGCGTACAG-3′ for β-actin; and 5′-CACCGCAATGGCTCCTTT-3′ and 5′-CACCAACTCTAGCAGCACATC-3′ for TIPE2.
10
Multiplex HRM for Species Identification
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Multiplex real-time PCR with HRM (MHRM) were done sequentially on a ABI StepOnePlus Real-Time PCR (Applied Biosystems) in a reaction mix containing 4 μl of 5x HOT FIREPol® EvaGreen® HRM Mix no ROX (Soils Biodyne, Estonia), 0.5 μl of each primer (10 pmol, Metabion, Martinsried, Germany), 1 μl of DNA (10–20 ng/μl) and 6.5 μl of DNase-free water in a total reaction volume of 20 μl per sample. Positive controls (genomic DNA from each species) and negative controls (distilled water) were used in each run. The reaction conditions were enzyme activation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for15 s, 63°C for 20 s for annealing and 72°C for 20 s for extension. Following this, HRM was performed at 95°C for 15 s and a melting profile from 65°C to 95°C using a ramping degree of 0.3°C/s. Then, the melt curve analysis was performed by HRM Software version 3.0.1 (Applied Biosystems). All samples were analyzed at least in triplicate.
The specificity of the HRM assay was evaluated using different DNA from other organisms such as; bacterial DNA of S. aureus, E. faecalis, Streptococcus pyogenes, Proteus mirabilis and Salmonella Typhimurium. Moreover, DNA from urogenital flora including Lactobacillus spp., S. epidermidis, yeast, viridans and nonhemolytic streptococci was also included in the assay.
The specificity of the HRM assay was evaluated using different DNA from other organisms such as; bacterial DNA of S. aureus, E. faecalis, Streptococcus pyogenes, Proteus mirabilis and Salmonella Typhimurium. Moreover, DNA from urogenital flora including Lactobacillus spp., S. epidermidis, yeast, viridans and nonhemolytic streptococci was also included in the assay.
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