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Stepone 7500 thermocycler

Manufactured by Thermo Fisher Scientific
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The StepOne 7500 thermocycler is a real-time PCR system designed for accurate and reliable nucleic acid amplification. It features a 96-well sample block, a sensitive optical detection system, and intuitive software for efficient experimental setup and data analysis.

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Lab products found in correlation

Miscript 2 rt kit, by Qiagen (2 mentions) Mirneasy micro kit, by Qiagen (2 mentions) Sybr green master mix, by Qiagen (1 mentions) First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

StepOne (326 citations) Thermocycler (175 citations) StepOne thermocycler (73 citations) 7500 thermocycler (28 citations)

4 protocols using stepone 7500 thermocycler

1

miRNA and mRNA Expression Analysis

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Five hundred nanograms of total RNA was used for first strand cDNA synthesis using the miScript RT II kit (Qiagen) according to the manufacturer’s instructions. Expression profiles of miR-223, miR-34a-5p and miR-142-3p were examined by miScript PCR assays and SYBR Green Master Mix (both from Qiagen). Real-time PCR was performed on a StepOne 7500 thermocycler (Applied Biosystems). Expression levels were normalized with respect to RNU6 (Qiagen). PKCα mRNA levels were analyzed using a StepOnePlus Real-Time PCR System (Applied Biosciences). Expression levels were normalized with respect to GAPDH.
Fordham J.B., Guilfoyle K., Naqvi A.R, & Nares S. (2016). MiR-142-3p is a RANKL-dependent inducer of cell death in osteoclasts. Scientific Reports, 6, 24980.
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2

Quantification of Renilla, Firefly, and miRNA

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For renilla and firefly mRNA quantification, 500 ng total RNA was reverse transcribed using the Superscript RT-II kit (Life Technologies, Waltham, MA, USA). A 20 μL reaction mix was prepared using 2X EvaGreen Master Mix (Biotium, Hayward, CA, USA), ~20 ng of cDNA, and 10 pmoles of each forward and reverse primer. The real-time PCR was carried out in a StepOne 7500 Thermocycler (Applied Biosystems, Carlsbad, CA, USA). GAPDH served as an internal control and all reactions were run in triplicates. The Ct values of replicates were analyzed to calculate relative fold change using the delta-delta Ct method. The data are presented as ratio of GAPDH normalized renilla and firefly mRNA expression. For mature miRNA (let-7a) quantification, let-7a miScript primers and miScript II RT Kit were purchased from Qiagen. One hundred nanograms of total RNA was reverse transcribed according to the manufacturer’s instructions. The reactions were run using miRNA specific primers and universal primer in the PCR mix buffer. RNU6 was used as endogenous control.
Kulkarni V., Naqvi A.R., Uttamani J.R, & Nares S. (2016). MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines. International Journal of Molecular Sciences, 17(1), 72.
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3

miRNA Regulation of Phagocytosis Genes

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Mφ and DC transfected with miRNA mimic or inhibitor were harvested after 36h. Total RNA was isolated using the miRNeasy micro kit (Qiagen) according to manufacturer's instructions. First strand cDNA was synthesized from 500 ng total RNA using the RT kit (Qiagen). A custom PCR array plate (96-well) containing 88 different genes involved in phagocytosis was used to assess gene expression (Qiagen). One microgram of cDNA was aliquoted onto each well and real-time PCR performed using a StepOne 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA). Expression levels were normalized with respect to beta-2 microglobulin as it demonstrated the most consistent levels across all transfections. Next, the fold change was calculated with respect to the negative miRNA control. Finally, the ratio of miRNA inhibitor and miRNA mimic was calculated. Real-time PCR for three randomly selected genes were also carried out in two independent donors.
Total RNA was isolated from E. coli challenged Mφ and DC and the expression of proinflammatory cytokine mRNA analyzed by RT-PCR. To monitor PKCα mRNA levels, total RNA was also isolated from monocytes, differentiating Mφ and DC at various time points. The data was presented as normalized fold change with standard deviation.
Naqvi A.R., Fordham J.B, & Nares S. (2015). miR-24, miR-30b and miR-142-3p regulate phagocytosis in myeloid inflammatory cells. Journal of immunology (Baltimore, Md. : 1950), 194(4), 1916-1927.
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4

RNA Isolation and Gene Expression Analysis

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Total RNA was isolated from 18 h, day 3, day 5, and day 7 differentiated cells using miRNeasy micro kit (Qiagen). A total of 250 ng RNA was used to synthesize cDNA, which was synthesized from first-strand cDNA synthesis kit (Invitrogen). The expression levels of GAS5, IPW, and β-actin genes were analyzed in a StepOne 7500 thermocycler (Applied Biosystems). The Ct values of three replicates were analyzed to calculate fold change using the 2−ΔΔCt method.
Ahmad I., Valverde A., Naqvi R.A, & Naqvi A.R. (2020). Long Non-coding RNAs RN7SK and GAS5 Regulate Macrophage Polarization and Innate Immune Responses. Frontiers in Immunology, 11, 604981.
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