Dataassist

Manufactured by Thermo Fisher Scientific

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DataAssist is a software tool designed for data analysis and visualization. It provides users with the ability to import, manage, and analyze various types of data. The software offers a range of features to facilitate data processing, interpretation, and presentation.

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19 protocols using dataassist

Quantitative miRNA expression analysis

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Additional samples were collected for validation and RNA was isolated as mentioned above using the miRNeasy kit (Qiagen). RNA (1 ng) was reverse transcribed to cDNA (TaqMan MicroRNA Reverse Transcription Kit, Applied Biosystems, Australia) according to the manufacturer’s protocol with a primer pool containing all miRNA assays (TaqMan microRNA assays, 5×, Applied Biosystems). Sixteen assays, which included 12 targets and 2 additional assays (hsa-miR-144–3p, hsa-miR–19b–3p, see Supplementary Table 6 for assay IDs), were pooled in order to serve as possible normalization controls. The cDNA samples were pre-amplified (TaqMan PreAmp Master Mix Kit, Applied Biosystems) and qRT–PCR (TaqMan Fast Advanced Master Mix, Applied Biosystems) was performed using individual miRNA assays (TaqMan microRNA assays, 20×, Applied Biosystems) and run on the ViiA 7 Real-Time PCR System (Life Technologies). Reverse transcription and pre-amplification no template controls using primer pools and individual assays were also prepared to ensure there was no background amplification of miRNA assays. Raw Ct data was uploaded to DataAssist (Applied Biosystems) to calculate delta delta Ct (ΔΔCt) using appropriate normalization methods^{68 (link)} calculated by DataAssist by Applied Biosystems.

Sisquella X., Ofir-Birin Y., Pimentel M.A., Cheng L., Abou Karam P., Sampaio N.G., Penington J.S., Connolly D., Giladi T., Scicluna B.J., Sharples R.A., Waltmann A., Avni D., Schwartz E., Schofield L., Porat Z., Hansen D.S., Papenfuss A.T., Eriksson E.M., Gerlic M., Hill A.F., Bowie A.G, & Regev-Rudzki N. (2017). Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors. Nature Communications, 8, 1985.

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miRNA Expression Profiling in PBMCs

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The miRNA expression profiles were determined with a TaqMan Array Human microRNA Card A (Thermo Fisher Scientific, Waltham, MA, USA). Ninety nanograms of total RNA was used as an input in each RT reaction. The RT reaction and pre-amplification step was set up according to the manufacturer’s recommendations. miRNAs were reverse transcribed with the Megaplex Primer Pools (Human Pools A version 2.1; Thermo Fisher Scientific). RT reaction products from the PBMC samples were further amplified with Megaplex PreAmp primers (Primers A version 2.1; Thermo Fisher Scientific). Quantitative RT-PCR was performed on an Applied Biosystems 7900HT thermocycler according to the manufacturer’s recommended program. With the use of SDS 2.4 software and DataAssist (Thermo Fisher Scientific), the expression of miRNA was calculated based on cycle threshold (Ct) values normalized by those of RNU6B. Data analysis was done using GeneSifter^® software (Perkin Elmer, Waltham, MA, USA). P-values of less than 0.05 were considered to indicate a statistically significant difference, and the Benjamini–Hochberg algorithm was used for estimation of false discovery rates, as we have reported previously.19 (link)

Ohyashiki J.H., Ohtsuki K., Mizoguchi I., Yoshimoto T., Katagiri S., Umezu T, & Ohyashiki K. (2014). Downregulated microRNA-148b in circulating PBMCs in chronic myeloid leukemia patients with undetectable minimal residual disease: a possible biomarker to discontinue imatinib safely. Drug Design, Development and Therapy, 8, 1151-1159.

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Quantitative Analysis of Gene Expression

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Real-time polymerase chain reaction (qPCR) was performed with commercial TaqMan^® Gene Expression Assay kits. In the qPCR reaction, commercial TaqMan^® Gene Expression Assay kits (Thermo Fisher Scientific Inc.) were used for the following genes: the members of the class O forehead box transcription factors (FOXO3s, Hs00818121_m1), mitogen-activated protein kinase 1 (MAPK1, Hs01046830_m1), and glucuronidase beta (GUSB, Hs00939627_m1) as potential endogenous controls. Assays were performed in 96-well optical plates using a 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific Inc.). For each qPCR reaction, 50 ng of cDNA, 0.5 µL of the appropriate TaqMan Gene Expression Assay, and 5 µL of TaqMan Universal Master Mix (Thermo Fisher Scientific Inc.) were used. The reaction was carried out in the volume of 10 μL under the following conditions: initial denaturation for 20 s at 95 °C and 40 cycles for 3 s at 95 °C and 30 s at 60 °C. The results were analyzed using the Sequence Detection System 2.4 software and Data Assist (Thermo Fisher Scientific Inc.) and presented as Cq or were calculated via ΔΔCt method values of the relative quantification of the expression (RQ).

Kolary-Siekierska K., Niewiadomski P., Namieciński W, & Miłoński J. (2023). Title Expression of FOXO3 and MAPK1 Genes in Patients with Benign Salivary Gland Tumors. Journal of Clinical Medicine, 13(1), 215.

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Transcriptional Analysis of Cellular Response to 1

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To evaluate the effect of 1 on various cell lines, cells were plated with RPMI or DMEM media containing 2% (v/v) FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin. The following morning the media was replenished with 0.1% DMSO and varying concentrations of 1. 48 hr after treatment with 1/DMSO the cells were harvested and RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN). cDNA was prepared using 1000 ng of purified RNA and the high capacity RNA to cDNA kit (ThermoFisher). 10 ng of cDNA was used for a 20 mL RT-PCR assay composed of Taqman Multiplex Master Mix (ThermoFisher) and the following Taqman probes for CTGF (Hs00170014 m1), CYR61 (Hs00155479 m1), TEAD1 (Hs00173359 m1), TEAD2 (Hs01055894 m1), TEAD3 (Hs00243231 m1), TEAD4 (Hs01125032 m1), YAP1 (Hs00902712 g1), WWTR1 (Hs00210007 m1), and GAPDH (Hs02786624 g1). RT-PCR assays were run using a Quantstudio 5. Relative expression was analyzed using DataAssist (ThermoFisher Scientific) and for three technical replicates for each biological replicate (n = 3).

Holden J.K., Crawford J.J., Noland C.L., Schmidt S., Zbieg J.R., Lacap J.A., Zang R., Miller G.M., Zhang Y., Beroza P., Reja R., Lee W., Tom J.Y.K., Fong R., Steffek M., Clausen S., Hagenbeek T.J., Hu T., Zhou Z., Shen H.C, & Cunningham C.N. (2020). Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling. Cell reports, 31(12).

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Quantitative Analysis of Gene Expression

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Total RNA was isolated from HEK293T cells, MEFs, and cultured keratinocytes from patient 1 and different control subjects by using the RNeasy Plus Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions. RNA samples were reverse transcribed into cDNA by using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). Real-time PCR was carried out with the Fast SYBR Green PCR Master Mix (Applied Biosystems) on an ABI Prism 7000 (PE Applied Biosystems). Quantitative RT-PCR primers were designed by using the sequences available in Ensembl and spanned an intron-exon boundary.
Amounts of the various mRNAs were normalized against the amount of ACTB RNA in each sample (measured by using quantitative RT-PCR). Results were analyzed with DataAssist (version 3.01; Applied Biosystems), which uses the comparative cycle threshold (ddCt) method. All experiments were performed in triplicates.
All data are expressed as the means ±SDs. Values were calculated by using an unpaired t test as follows.

Polivka L., Hadj-Rabia S., Bal E., Leclerc-Mercier S., Madrange M., Hamel Y., Bonnet D., Mallet S., Lepidi H., Ovaert C., Barbet P., Dupont C., Neven B., Munnich A., Godsel L.M., Campeotto F., Weil R., Laplantine E., Marchetto S., Borg J.P., Weis W.I., Casanova J.L., Puel A., Green K.J., Bodemer C, & Smahi A. (2018). Epithelial barrier dysfunction in desmoglein-1 deficiency. The Journal of allergy and clinical immunology, 142(2), 702-706.e7.

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Statistical Analysis of Epilepsy Expression Data

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All statistical analyses were performed using the Prism GraphPad, version 8.0 (GraphPad Software, Inc., CA, US). The relationship between categorical variables was compared using the χ² test. Further analysis was performed in DataAssist (Applied Biosystems, CA, USA). The p-value was calculated based on a two-sample, two-tailed Student’s t-test for the calculated. Fold change (relative to the epilepsy group) and a p-value were generated on a two-sample, two-tailed Student’s t-test. One Matrix CIM online package (https://discover.nci.nih.gov/cimminer/home.do) was used for drawing the heatmap and hierarchical clustering. Expression analysis data were taken in three replication and given as mean value ± SE.

Dehbashi S., Tahmasebi H., Zeyni B, & Arabestani M.R. (2021). Regulation of virulence and β-lactamase gene expression in Staphylococcus aureus isolates: cooperation of two-component systems in bloodstream superbugs. BMC Microbiology, 21, 192.

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Intestinal Epithelial Cell RNA Isolation

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cDNA was generated from DNase treated RNA harvested from intestinal epithelial cells isolated from the whole small intestine (E16.5–E18.5) or from intact jejunal tissue harvested from the midpoint of small intestine (E15.5) as previously described (Duncan et al., 1997 (link); Bondow et al., 2012 ). Supplemental Tables 1 and 2 contain primer sequences and TaqMan assay identifiers, respectively. qRT-PCR data were analyzed using DataAssist software (Applied Biosystems, Carlsbad, CA). Gapdh was used for normalization. Each gene was assayed in at least three independent experiments using cDNA from three control and three mutant intestines. Error bars represent standard error of the mean (SEM).

Walker E.M., Thompson C.A, & Battle M.A. (2014). GATA4 and GATA6 regulate intestinal epithelial cytodifferentiation during development. Developmental biology, 392(2), 283-294.

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Quantitative Real-time PCR Gene Expression Analysis

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Total RNA was extracted, reverse transcribed, and used for real-time qPCR experiments as described by Nonis et al. (2012) (link). Selective primers were constructed on divergent putative 3’-untranslated regions (UTRs) determined by sequence alignments and poly(A)-tail prediction (HCpolyA; Milanesi et al., 1996 (link)). Primers (Supplementary Table S1, available at JXB online) were designed with Primer3 (Rozen and Skaletsky 2000 (link)) and tested with PRaTo (Nonis et al., 2011 (link)). Data were elaborated with DataAssist (Applied Biosystems, Monza, Italy) and normalized to Md_8283:1:a (Botton et al., 2011 (link)) using the Livak and Schmittgen (2001) (link) method. General good-practice guidelines for RT-qPCR (Udvardi et al., 2008 (link); Remans et al., 2014 (link)) were adopted and primers efficiencies (Supplementary Table S1) were calculated and considered for differentially expressed genes as described by Nonis et al. (2012) (link). The choice of RT-qPCR for gene expression analyses was based on suggestions reported by Nonis et al. (2014) (link). All analyses were carried out on three independent biological replicates for each time point and experimental condition.

Zermiani M., Zonin E., Nonis A., Begheldo M., Ceccato L., Vezzaro A., Baldan B., Trentin A., Masi A., Pegoraro M., Fadanelli L., Teale W., Palme K., Quintieri L, & Ruperti B. (2015). Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits. Journal of Experimental Botany, 66(22), 7255-7270.

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Repeated Measures ANOVA in Alcohol Consumption

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Repeated measures analyses of variance (ANOVAs) were used to analyze the consumption along the drinking sessions for animals that completed all sessions (i.e., Familiar II group). One way ANOVAs were performed to compare consumption of the different groups. Expression data analyses were performed using DataAssist software (Applied Biosystems, version 3.01). Relative Quantification (RQ) values (relative levels of RNA expression) were calculated using the comparative Ct method with endogenous controls to normalize the data. Extreme values ranging more than two standard deviations were removed from the sample as that might create artificial baseline levels of gene expression. Before analysis, the data were tested for distribution and found to be normally distributed. Repeated measures analyses of variance (ANOVAs) were used to compare each gene expression in each brain zone. Post hoc Fisher LSD test comparisons between the groups were used. Differences were considered as statistically significant at p < 0.05.

Gómez-Chacón B., Gámiz F., Foster T.C, & Gallo M. (2015). Increased N-Ethylmaleimide-Sensitive Factor Expression in Amygdala and Perirhinal Cortex during Habituation of Taste Neophobia. Neural Plasticity, 2016, 2726745.

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Unpaired, Non-parametric t-Test Analysis

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Statistical analysis was performed using Prism 6 (Graph-Pad Software Inc., San Diego, California, USA). Differences between groups were assessed using an unpaired, non-parametric, two-tailed t test. A p value ≤0.05 was considered statistically significant. The relative expression and the error bars for the 2^−ΔΔCt method were calculated using Data Assist (Applied Biosystems).

Mathios D., Ruzevick J., Jackson C.M., Xu H., Shah S., Taube J.M., Burger P.C., McCarthy E.F., Quinones-Hinojosa A., Pardoll D.M, & Lim M. (2014). PD-1, PD-L1, PD-L2 expression in the chordoma microenvironment. Journal of neuro-oncology, 121(2), 251-259.

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