Expression suite software 1

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Expression Suite Software 1.0.3 is a data analysis software developed by Thermo Fisher Scientific. The software is designed to process and analyze gene expression data from various sources. It provides tools for data normalization, visualization, and statistical analysis.

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9 protocols using expression suite software 1

Quantification and Analysis of Pancreatic Islet RNA

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Total RNA extracted from EndoC-βH1 and collagenase-isolated human pancreatic islet samples were quantified using Qubit 3000 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA), while those extracted from LCM-islets were quantified using 2100 Bioanalyzer-RNA 6000 Pico Kit (cat. 50671513, Agilent Technologies, Santa Clara, CA, USA) as well as RNA integrity (RIN). Samples with RIN<5.0 were excluded. Reverse transcriptase reaction was performed using SuperScript™ VILO™ cDNA Synthesis Kit (cat. 11754050-ThermoFisher Scientific, Waltham, MA, USA).
cDNA derived from LCM human pancreatic islets was then amplified using TaqMan PreAmp Master Mix (cat. 4488593, ThermoFisher Scientific, Waltham, MA, USA) following manufacturer’s instructions.
Real-Time PCR analysis was performed using TaqMan gene expression assays using primers (see Resources Table) and SensiFast Probe Lo-ROX Kit (cat.# BIO-84020, Bioline) following manufacturer’s recommendation. Data were collected and analyzed through Expression Suite software 1.0.1 (ThermoFisher Scientific, Waltham, MA, USA) using 2^−ΔCt or 2^−ΔΔCt method. ViiA7 Real-Time PCR thermalcycler instrument (ThermoFisher Scientific, Waltham, MA, USA) was used to perform Real-Time PCR reactions.

Fignani D., Licata G., Brusco N., Nigi L., Grieco G.E., Marselli L., Overbergh L., Gysemans C., Colli M.L., Marchetti P., Mathieu C., Eizirik D.L., Sebastiani G, & Dotta F. (2020). SARS-CoV-2 Receptor Angiotensin I-Converting Enzyme Type 2 (ACE2) Is Expressed in Human Pancreatic β-Cells and in the Human Pancreas Microvasculature. Frontiers in Endocrinology, 11, 596898.

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Quantifying miR-184-3p Promoter Enrichment

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Real-time PCR was performed to quantify the amplification of the immunoprecipitated miR-184-3p sequence promoter fragment. 1/30 of the recovered DNA was added to the PCR reaction, which consisted of 12.5 µL Maxima SYBR Green qPCR Master mix (2×), 0.05 µl ROX solution and nuclease-free H₂O. The following primers were used for the human MIR184 gene promoter (300 nM each): forward primer 5’-AATGGCATGTGGGTGTTGGT-3’; reverse primer 5’-AGGGCTCCTGCAGGTCTGA-3’. The following primers were used for miR-184-3p murine promoter (500 nM): forward primer 5’-AATGGCATGTGGGTGTTGGT-3’; reverse primer 5’-AGGGCTCCTGCAGGTCTGA-3’.
The reaction was incubated at 95 °C for 10 min, 95 °C for 15 s – 60 °C for 30 s – 72 °C for 30 s (40 cycles) in ViiA7 Real-time PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA). Data were collected and analysed with Expression Suite software 1.0.1 (Thermo Fisher Scientific, Waltham, MA, USA) using 2^−ΔCt method. The raw Ct data resulting from chromatin immunoprecipitated with specific antibodies were normalised to the negative isotypic control rabbit IgG (cat. 492024, Thermo Fisher Scientific, Waltham, MA, USA). Statistical significance was calculated using a paired parametric two-tailed t-test (on fold change) with a significance cutoff p < 0.05.

Grieco G.E., Brusco N., Fignani D., Nigi L., Formichi C., Licata G., Marselli L., Marchetti P., Salvini L., Tinti L., Po A., Ferretti E., Sebastiani G, & Dotta F. (2022). Reduced miR-184-3p expression protects pancreatic β-cells from lipotoxic and proinflammatory apoptosis in type 2 diabetes via CRTC1 upregulation. Cell Death Discovery, 8, 340.

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Relative Gene Expression Quantification

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Relative gene expression values were calculated with 2^−Ct (ref. 69 (link)) using Expression Suite Software 1.0.3 (Life Technologies). RQ minimum and maximum values (error bars) were calculated with a confidence level of 95%, using Benjamini-Hochberg false discovery rate to adjust P values. Maximum allowed Ct included in calculations is 34 and Cq confidence > 0.8. Multivariate Student’s t-test was applied and values of P < 0.05 were considered statistically significant. Error bars are presented in all graphs as s.d. Gene expression profile data are represented in graphics as relative quantity of 2^−Ct of 21Q normalized to others.

Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice. (2017). Nature neuroscience, 20(5), 648-660.

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Relative Gene Expression Quantification

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Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice. (2017). Nature neuroscience, 20(5), 648-660.

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RNA Extraction and qPCR Analysis

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RNA was extracted using the RNeasy kit (Qiagen; Germantown, MD) following the manufacturer’s instructions and quantified using the NanoDrop 1000 spectrophotometer (Thermo Scientific; Wilmington, DE). First-strand cDNA was synthesized from 500 ng total RNA per reaction using the TaqMan RNA-to-Ct 1-Step kit (Life Technologies; Grand Island, NY) according to the manufacturer’s protocol, followed by qPCR analysis in conjunction with the ABI Prism 7900 HT Sequence Detector apparatus (Life Technologies). Cycle threshold value was generated using SDS software version 2.3 (Life Technologies). The −ΔΔCt method was used to determine fold-change of gene expression between samples. Data were normalized using beta-actin (ACTB) and then analyzed using ExpressionSuite Software 1.0.3 (Life Technologies). A two-tailed t-test was used to determine p-values.

Hanson R.L., Leti F., Tsinajinnie D., Kobes S., Puppala S., Curran J.E., Almasy L., Lehman D.M., Blangero J., Duggirala R, & DiStefano J.K. (2016). The Arg59Trp variant in ANGPTL8 (Betatrophin) is associated with total and HDL-cholesterol in American Indians and Mexican Americans and differentially affects cleavage of ANGPTL3. Molecular genetics and metabolism, 118(2), 128-137.

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Quantitative Gene Expression Analysis

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Total cellular RNA was isolated by the modified method of Chomczyński [63 (link)] from the obtained cells using TRI Reagent (Sigma), isopropanol (Sigma), chloroform (Sigma), ethyl alcohol (Poch, Poland). The RNA extract was spectrophotometrically evaluated.
The reverse transcription reaction was carried out as recommended by the manufacturer, using the High-Capacity reagent cDNA Transcription Kits with RNase Inhibitor (Applied Biosystems, USA) and 1 μg of isolated RNA.
Relative gene expression levels of NAIP, BIRC2, BIRC3, BIRC5, BIRC6, BIRC7, BIRC8, XIAP, XAF1, OCT4 and SOX2 were examined by the qPCR method using commercially available TaqMan probes (Applied Biosystems, USA): GAPDH: Hs99999905_m1; for NAIP gene: Hs03037952_m1; for BIRC2 gene: Hs00357350_m1; for BIRC3 test gene: Hs00154109_m1; for BIRC5: Hs00153353_m1; for BIRC6: Hs00212288_m1; for BIRC7: Hs01086675_m1; for BIRC8: Hs01057786_s1; for XIAP: Hs00236913_m1; for XAF1: Hs00213882_m1; for OCT4: Hs00242302_m1; and for SOX2 gene: Hs00193931_m1. GAPDH was an endogenous control gene. The expression of the examined genes was calculated from the formula RQ = 2^−ΔΔCT [64 (link)]. The Expression Suite Software 1.0.3 was used to calculate gene expression level (Life Technologies). Detailed procedures for the RNA isolation and qPCR reaction shave have been described in our previous work [23 (link),48 (link)].

Gil-Kulik P., Leśniewski M., Bieńko K., Wójcik M., Więckowska M., Przywara D., Petniak A., Kondracka A., Świstowska M., Szymanowski R., Wilińska A., Wiliński M., Płachno B.J., Kostuch M., Rahnama-Hezavach M., Szuta M., Kwaśniewska A., Bogucka-Kocka A, & Kocki J. (2023). Influence of Perinatal Factors on Gene Expression of IAPs Family and Main Factors of Pluripotency: OCT4 and SOX2 in Human Breast Milk Stem Cells—A Preliminary Report. International Journal of Molecular Sciences, 24(3), 2476.

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Fetal Gene Expression Profiling

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Human WGE samples from 49 to 64 ultrasound pcd (postconceptional day) were consider as the WGE biological group in order to acquire more statistical power, with the caveat of losing biological information during this fetal development period. Relative gene expression values were calculated using Expression Suite Software 1.03 (Life Technologies, Barcelona, Spain). RQ minimum and maximum values (error bars) are calculated with a confidence level of 95%, using Benjamini-Hochberg false discovery rate to adjust P values. Maximum allowed C_t included in calculations is 30 and C_q confidence > 0.8. Multivariate Student’s t-test or one-way ANOVA were applied and values of P < 0.05 were considered statistically significant. Error bars are presented in all graphs as standard error of the mean (SEM). Gene expression profile data are represented in graphics as relative quantity or fold change of one biological group normalized to another. Volcano plot analysis comparing WGE and striatal samples was performed with Data Assist v3.01 software (Applied Biosystem) using the same parameters described above for the Expression Suite software.

Straccia M., Garcia-Diaz Barriga G., Sanders P., Bombau G., Carrere J., Mairal P.B., Vinh N.N., Yung S., Kelly C.M., Svendsen C.N., Kemp P.J., Arjomand J., Schoenfeld R.C., Alberch J., Allen N.D., Rosser A.E, & Canals J.M. (2015). Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons. Molecular Therapy. Methods & Clinical Development, 2, 15030-.

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Differential miRNA Expression Analysis

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For array data analysis, SDS Software v.2.3 (Thermo Fisher Scientific) was used to calculate Raw Ct values, with an automatic baseline and threshold. ExpressionSuite Software 1.1 (Thermo Fisher Scientific) was used to calculate RQ (2^-∆∆Ct) values. Data were normalized using global normalization, an algorithm that finds the assays common to every sample and then uses the median Ct of those assays as the normalization factor, on a per sample basis [25 (link)]. Ct values > 35 or with Amp score < 0.7 were excluded from the analysis. To identify candidate miRNAs differentially expressed between patients and controls, we selected miRNAs with low p values (p ≤ 0.05). P values were calculated by the ExpressionSuite software using Student’s t-test for sample group comparisons, without multiple test correction. Further to this, we screened the group for miRNAs expressed in every sample and with low variability among the same group.
For qRT-PCR data analysis, Excel software (Microsoft Office 365 ProPlus) was used to calculate ∆Ct, −∆∆Ct, and RQ for patients and controls. Statistical analysis was performed on RQ values through the demo version of GraphPad Prism 6.01 software using an unpaired non-parametric two-sided Mann-Whitney test. Confidence level was set at 95% (p value ≤0.05).

Gai C., Camussi F., Broccoletti R., Gambino A., Cabras M., Molinaro L., Carossa S., Camussi G, & Arduino P.G. (2018). Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma. BMC Cancer, 18, 439.

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Extracellular Vesicle miRNA Profiling

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miRNA expression analysis was performed by qRT-PCR array on three different samples of BMSC-EVs. Briefly, RNA was extracted from purified EVs by mirVana Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA), according with manufacturer’s instruction. RNA concentration was measured by Nanodrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA), and the ratio 260/280 and 260/230 showed absence of contaminants. Fifty nanograms of total RNA were retro-transcribed to cDNA with TaqMan^® MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was pre-amplified with Megaplex™ RT Primers, Human Pool Set v3.0 and TaqMan^® PreAmp Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) by using Veriti Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). The expression profile of a panel of 754 human miRNAs was evaluated by TaqMan^® Array Human MicroRNA Card Set v3.0 (Thermo Fisher Scientific, Waltham, MA, USA) with QuantStudio12k Flex system (Thermo Fisher Scientific, Waltham, MA, USA). ExpressionSuite Software 1.1 (Thermo Fisher Scientific, Waltham, MA, USA) was used to calculate Ct values. Values of Amp score < 1.1 or Cq conf < 0.7 or Ct > 34 were excluded from analysis. Only miRNAs expressed in all samples of the same group were included for analysis.

Pomatto M., Gai C., Negro F., Cedrino M., Grange C., Ceccotti E., Togliatto G., Collino F., Tapparo M., Figliolini F., Lopatina T., Brizzi M.F, & Camussi G. (2021). Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes. International Journal of Molecular Sciences, 22(8), 3851.

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