Stepone software version 2.3

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, France

StepOne Software (version 2.3) is a real-time PCR analysis software developed by Thermo Fisher Scientific. It provides a user interface for the analysis and management of real-time PCR experiments.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

StepOne (326 citations) StepOne software (286 citations) StepOne Software version 2.3 (131 citations) StepOne Software 2.0 (47 citations) StepOne 2.1 software (22 citations) StepOne, version 2.1 (11 citations)

6 protocols using stepone software version 2.3

Sensitive qPCR Detection of Mycobacterium avium

Check if the same lab product or an alternative is used in the 5 most similar protocols

Two already described qPCRs targeting IS900 (Donaghy et al. 2011) and f57 (Ricchi et al. 2014) sequences were employed in this study. f57 is present in only one copy in MAP genome, while IS900 is present in around 15–18 copies, which means the latter qPCR has potentially superior detection sensitivity. An internal positive control (TaqMan^® Exogenous Internal Positive Control, Life Technologies, Monza, Italy) was added to each PCR reaction, in order to detect possible false‐negative results due to PCR inhibition. A negative extraction sample was processed in parallel during each DNA extraction. These last samples, together with a negative and a positive PCR controls, were added to all qPCR runs in order to check each amplification reaction. All reactions were run on StepOne Plus qPCR system (Life Technologies) using TaqMan Universal Master Mix without UNG (Life Technologies). Raw data were processed by StepOne Software (version 2.3) (Life Technologies). According to minimum information for publication of quantitative real‐time PCR experiments (MIQE) guidelines (Bustin et al. 2009), information about the analytical sensitivity and efficiency of both qPCRs (IS900 and f57) and linear range are available in Figure S1, while information about the analytical specificity and reagents concentrations are reported in the original papers (Donaghy et al. 2011; Ricchi et al. 2014).

Ricchi M., Savi R., Bolzoni L., Pongolini S., Grant I.R., De Cicco C., Cerutti G., Cammi G., Garbarino C.A, & Arrigoni N. (2016). Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia‐Romagna Region (Italy) by qPCR. MicrobiologyOpen, 5(4), 551-559.

+ Open protocol

+ Expand

Quantitative RT-PCR Analysis of Zebrafish Embryos

Check if the same lab product or an alternative is used in the 5 most similar protocols

Pools of 20–25 embryos were frozen on dry ice at 24hpf. Embryos were homogenized in lysis buffer using a 23-gauge needle and extraction of RNA from the embryos was carried out using the RNAqueous 4-PCR kit (Ambion). Quantification of purified RNA was carried out using a Nanodrop spectrophotometer machine. Next, cDNA synthesis was performed using SuperScript^® VILO cDNA Synthesis Kit or Superscript III cDNA synthesis kit (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using PowerUp^™ SYBR^™ Green Master Mix (Applied Biosystems) in a StepOnePlus^™ Real-Time PCR System. Quantification was performed using the relative standard curve method; controls/DMSO treatment were assigned a value of 1 and experimental values were computed by the software based on the C_T values, relative to the control standard curve. For each experiment, 2–3 replicates were performed with duplicates in each run and ef1α was used as an endogenous control. The relative quantity of cDNA in each sample of each gene was normalized to the value of ef1α. Data was analyzed using StepOne^™ Software-version 2.3 (Life Technologies).
The following primers were used:

Baltrunaite K., Craig M.P., Desai S.P., Chaturvedi P., Pandey R.N., Hegde R.S, & Sumanas S. (2017). ETS transcription factors Etv2 and Fli1b are required for tumor angiogenesis. Angiogenesis, 20(3), 307-323.

+ Open protocol

+ Expand

Quantitative PCR for Gene Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

QPCR assays were performed using TaqMan® RNA-to-Ct™ 1-Step Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Pre-designed TaqMan assays for human albumin (targets, Hs00609411_m1 and Hs00910225_m1), GAPDH (endogenous control, Hs02786624_g1 and Hs99999905_m1), and AFP (target, Hs01040598_m1) were purchased from Life Technologies (Life Technologies, Carlsbad, CA, USA). Assay results were analyzed using Life Technologies StepOne Software Version 2.3. The expression level was calculated as the value of 40–delta C_t (C_t target–C_t endogenous control) and the fold change relative to HepG2 RNA was calculated using the formula: fold = 2–ΔΔCt (ΔΔCt = ΔCt test sample–ΔCt control sample). All results were then normalized to the lowest detectable RNA sample.

Chu X., Karasinski K., Donellan S., Kaniper S., Wood G.C., Shi W., Edwards M.A., Soans R., Still C.D, & Gerhard G.S. (2019). A retrospective case control study identifies peripheral blood mononuclear cell albumin RNA expression as a biomarker for non-alcoholic fatty liver disease. Langenbeck's archives of surgery, 405(2), 165-172.

+ Open protocol

+ Expand

Quantifying Bacterial Diversity via qPCR

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Quantitative real-time PCR was carried out for quantification of 16S rRNA gene copy number in order to determine the volume of Preservcyt required for sequencing and to compare the bacterial load collected by each technique in total and of G. vaginalis. Real-time qPCR was performed with universal BactQUANT 16S rRNA gene primers (Forward primer: 5′-CCTACGGGAGGCAGCA, Reverse primer: 5′-GGACTACCGGGTATCTAATC) (Sigma) with the FAM labeled BactQUANT probe ((6FAM) 5′-CAGCAGCCGCGGTA-3′ (MGBNFQ))^{30 (link)} and G. vaginalis primers (Forward primer: 5′-GGAAACGGGTGGTAATGCTGG, Reverse primer: 5′-CGAAGCCTAGGTGGGCCATT)^{31 (link)} using a SYBR green-based assay on the Applied Biosciences StepOne machine (Thermo Fisher Scientific, Ashford, UK) with StepOne software version 2.3 (Life Technologies). Samples were run in duplicate.

Mitra A., MacIntyre D.A., Mahajan V., Lee Y.S., Smith A., Marchesi J.R., Lyons D., Bennett P.R, & Kyrgiou M. (2017). Comparison of vaginal microbiota sampling techniques: cytobrush versus swab. Scientific Reports, 7, 9802.

+ Open protocol

+ Expand

Quantitative Real-time PCR Analysis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

In addition to conventional PCR, quantitative real-time PCR was performed. Therefore, Step One Plus^® Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) and GreenMasterMix, High ROX (Genaxxon Bioscience, Ulm, Germany) were used [19 (link)]. The same primer and DNA concentrations used for conventional PCR were used here. qPCR was carried out with the following parameters: 15 min at 95 °C for initial denaturation; 40 cycles of amplification with the following steps: denaturation for 15 s at 95 °C, annealing for 30 s at 62 °C, and extension for 30 s at 72 °C. Evaluation of the results was performed using StepOne Software version 2.3 (Life Technologies).

Ruoß M., Kieber V., Rebholz S., Linnemann C., Rinderknecht H., Häussling V., Häcker M., Olde Damink L.H., Ehnert S, & Nussler A.K. (2019). Cell-Type-Specific Quantification of a Scaffold-Based 3D Liver Co-Culture. Methods and Protocols, 3(1), 1.

+ Open protocol

+ Expand

Quantitative RT-PCR Analysis of Gene Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA extraction and quantitative RT-PCRs (QRT-PCRs) were performed as previously described [6 (link)]. Briefly, LS174T cells were washed three times with ice-cold PBS and homogenized in Tri-Reagent (Euromedex, Souffelweyersheim, France). Total RNA (1 µg) was next reverse transcribed using the High-Capacity RNA-to-cDNA^TM kit (Applied Biosystems, France). Q-PCRs were performed using the StepOne Plus real-time PCR system (Applied Biosystems, France) and were carried out using the following human specific primers synthesized by Eurogentec Company (Angers, France): Muc2 (F-5’CAG CAC CGA TTG CTG AGT TG3’, R-5’GCT GGT CAT CTC AAT GGC AG3’), KLF4 (F-5’AGA GGA GCC CAA GCC AAA GA3’, R-5’CAG TCA CAG TGG TAA GGT TTC TC3’), glucose-related protein 78 kDa (GRP78) (F-5’TGC TGC TAG GCC TGC TCC GA3’, R-5’CGA CCA CCG TGC CCA CAT CC3’), CHOP (F-5’CTG CCT TTC ACC TTG GAG AC3’, R-5’ CGT TTC CTG GGG ATG AGA TA 3’), ATF4 (F-5’ATG GCC GGC TAT GGA TGA T3’, R-5’CGA AGT CAA ACT CTT TCA GAT CCA TT3’), and β-actin (F-5’ATG ATA TCG CCG GGC TCG TCG TC3’, R-5’AGG TCC CGG CCA GCC AGG TCCAG3’). Finally, threshold cycle values were calculated by using Step One Software version 2.3 (Life technologies, Illkirch, France). Expression levels of target genes were normalized with the housekeeping gene β-actin and the 2^−ΔΔCt method was used to compare the relative expression of gene expression.

Escoula Q., Bellenger S., Narce M, & Bellenger J. (2019). Docosahexaenoic and Eicosapentaenoic Acids Prevent Altered-Muc2 Secretion Induced by Palmitic Acid by Alleviating Endoplasmic Reticulum Stress in LS174T Goblet Cells. Nutrients, 11(9), 2179.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!