cDNA was prepared as describe above and qRT-PCR was performed on an ABI StepOnePlus™ Real-Time PCR machine (Applied Biosystems, Foster City, CA) using a Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Generation of a single gene-specific PCR product was confirmed by melting curve analysis. Relative expression was calculated using the comparative Ct method as described previously33 (link). All PCR reaction samples were prepared in triplicate for each gene. The relative mRNA expression levels were determined by. The primer sequences were as follows: ADM, forward 5′-TGCCCAGACCCTTATTCGG-3′, reverse 5′-AGTTGTTCATGCTCTGGCGG-3′.
Abi steponeplus real time pcr machine
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
The ABI StepOnePlus Real-Time PCR machine is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qPCR) analysis. It is capable of monitoring and quantifying nucleic acid amplification in real-time.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Illustra rnaspin mini kit, by GE Healthcare (2 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Reverse transcription reagent, by Toyobo (2 mentions)
Sybr green real time pcr master mix kit, by Toyobo (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Revertaid mmlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green, by Thermo Fisher Scientific (1 mentions)
Sybr pcr master mix, by Thermo Fisher Scientific (1 mentions)
Anchored oligo dt primers, by Thermo Fisher Scientific (1 mentions)
Sensifast sybr hi rox kit, by Meridian Bioscience (1 mentions)
Micro kit, by Qiagen (1 mentions)
Rneasy mini, by Qiagen (1 mentions)
Tempus spin rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Sybr green reagent, by Takara Bio (1 mentions)
23 protocols using abi steponeplus real time pcr machine
1
Quantitative Real-Time PCR for ADM Gene
2
Quantitative real-time PCR analysis
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Real time PCR was performed to quantify the expression of selected DEGs (Table S5 ) obtained from Illumina sequencing. The cDNAs, used as template, were generated from mRNA using the REVERTAID MMLV reverse transcriptase (Fermentas). Primers (Table S6 ) were designed using the primer designing tool at IDT (https://eu.idtdna.com/site ) to amplify an amplicon of 80–150 nucleotides with Tm around 60 °C. Reactions were run in triplicates (technical and biological) for each sample using Power-Up SYBR Green on an ABI StepOnePlus real time PCR machine (Applied Biosystems Inc, USA) and the data analyzed was the mean of biological triplicates. The reaction was set up in 20 µl as follows: 1 µl of cDNA, 10 µl SYBR Green Dye master mix (2X), 5 pmol each of forward and reverse primers and water up to 20 µl. The general steps performed during real-time PCR experiment were as follows: step 1, 50 °C, 2 min, step 2, 95 °C, 10 min, step 3 (95 °C 15 s, 60 °C 1 min) × 40 cycles. The specificity of the amplicon was analyzed by a melt curve analysis. The relative mRNA level of the gene in different RNA samples was normalized with respect to ACTIN as the internal control gene60 (link) and analyzed by 2−∆CT method133 (link).
3
Quantitative RT-PCR Gene Expression Analysis
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For quantitative Reverse-Transcriptase PCR assays, we extracted the total mRNA from the cells using an illustra RNAspin Mini kit (GE Healthcare) according to the manufacturer’s instructions. Extracted RNA was subjected to cDNA synthesis using a High-Capacity cDNA reverse transcription kit (Applied Biosystems, USA). Each PCR reaction consisted of 10 μl of 2X SYBR PCR master mix (Applied Biosystems), 1μM of each forward and reverse primers and 2μl of the cDNA. The cDNA was amplified on an ABI StepOne plus real-time PCR machine (Applied Biosystems), and the relative gene copies or the transcripts were calculated with the ΔΔCT method. Each experiment included duplicate samples and the data shown represent the mean of three independent experiments. We calculated P values using two-tailed t-tests with Graphpad (Prism 6, USA) software.
4
Quantitative Real-Time PCR Analysis
5
Quantitative Real-Time PCR Analysis of Gene Expression
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The cells (2 × 105) were seeded in a 24-well plate until reaching 60–70% confluence, and then treated with 50 or 100 μg/ml PSA for 6, 12, and 24 h depending on the experiment. RNA was isolated using TRIZOL reagent (Ambion, United States) and converted to cDNA using reverse transcription reagents (TOYOBO, Japan) according to the manufacturer’s protocols. For determination of mRNA expression, RT-PCR was performed using SYBR Green Real time PCR Master Mix Kit (TOYOBO, Japan). The reaction was operated with an ABI StepOne plus real-time PCR machine (Applied BiosystemsTM, United States) under the following conditions: initial heat activation at 95°C for 1 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s (40 cycles). Primer sequences are listed in Table 1 .
6
Total RNA Extraction and qRT-PCR Analysis
7
SARS-CoV-2 Transcript Profiling in Blood
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Total RNA from Tempus blood was extracted using the Tempus Spin RNA isolation kit (Applied Biosystems, 4380204). cDNA was obtained using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR (qPCR) was performed using the TaqMan Fast Advanced Master Mix (Applied Biosystems) on the ABI StepOnePlus Real-Time PCR machine (Applied Biosystems). The following cycling conditions were used: 95 °C for 2 min; followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. IFI27 and GAPDH were amplified using the TaqMan Gene Expression Assay probes Hs01086373_g1 (IFI27) and Hs02786624_g1 (GAPDH). GAPDH was used as a housekeeping gene control. The unexposed prepandemic control HCW cohort for qPCR analysis was described previously57 (link)
8
Quantitative RNA Expression Analysis
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Total RNA from tissues was isolated using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. cDNA was synthesized using the ReverTra Ace (Toyobo, Osaka, Japan). qRT-PCR was performed using the ABI StepOne Plus Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). The primers used in this study are listed in Table S1
9
Gene Expression Analysis by RT-PCR
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RNA was converted to cDNA using reverse transcription reagents (TOYOBO, Japan) according to the manufacturer's protocol. RT-PCR was performed using SYBR Green Real time PCR Master Mix Kit (TOYOBO, Japan) with primers listed in Table S3 . The reaction was performed with an ABI StepOne plus real-time PCR machine (Applied Biosystems™, CA) at the Soonchunhyang Biomedical Research Core Facility of Korea Basic Science Institute. Expression of target genes was calculated by comparison of relative levels after normalization to GAPDH.
10
Quantitative Analysis of Gene Expression
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Total RNA was used to synthesize the first strand cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser Perfect Real Time (Takara, Japan). RT-qPCR was conducted with SYBR Green reagents (Takara, Japan) using an ABI StepOne Plus real-time PCR machine (Applied Biosystems). The PCR program was as follows: 94°C for 5 min, 40 cycles of 30 s at 94°C, 30 s at 60°C and a final melting curve at 65−95°C. GADPH was used as internal reference gene to quantify cDNA. The threshold cycle (Ct) values of the PCR were averaged and the relative transcript levels were quantified using the 2–△△Ct method. All primers used for the RT-qPCR are listed in Supplementary Table 1 .
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