Abi 7500 sds software

Manufactured by Thermo Fisher Scientific

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The ABI 7500 SDS software is a computer program designed to control and analyze data from the ABI 7500 Real-Time PCR System, a widely used instrument for quantitative real-time PCR analysis. The software enables users to set up, run, and analyze real-time PCR experiments with the ABI 7500 instrument.

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10 protocols using abi 7500 sds software

Quantitative RT-PCR for Calcitonin Receptor-Like Receptor Gene Expression

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Total cellular RNA was extracted using Trizol^¯ Reagent (Invitrogen, USA),
and RNA was reverse transcribed to single-stranded cDNA using a High Capacity Kit
(Applied Biosystems, USA) according to the manufacturer's protocol. For quantitative
analysis of the genes of interest [pre-pro-AM (Rn 00562327_m1), CRLR (Rn
00562334_m1), RAMP1 (Rn 01427056_m1), RAMP2 (Rn 00824652_m1), and RAMP3 (Rn
00571815_m1)], a commercially available TaqMan Assay-on-Demand System that consists
of a kit of oligonucleotides and probes was used (Applied Biosystems). Reverse
transcription was performed using 1 µg total RNA for each sample in 20 µL of the
total reaction mixture. The cDNA obtained was diluted 1:10, and 4.5 µL was used for
each 10 µL of the qRT-PCR mixture using the TaqMan Master Mix (Applied Biosystems).
Reactions were carried out in duplicate and analyzed with 7500 Sequence Detection
System apparatus (Applied Biosystems). Data were analyzed using the ABI-7500 SDS
software (Applied Biosystems). Total RNA absorbed was normalized on the basis of the
Ct value for the GAPDH gene (Rn 01775763_m1). The variation in expression among
samples was calculated by the 2^−ΔΔCt method, and the mean delta Ct value
for a group of six samples from the control was used for calibration (17 (link)).

Leite L.N., Gonzaga N.A., Tirapelli D.P., Tirapelli L.F, & Tirapelli C.R. (2014). Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle. Brazilian Journal of Medical and Biological Research, 47(10), 876-885.

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Quantitative RT-PCR Analysis of Gene Expression

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The total RNA of each sample was isolated with a total RNA isolation Kit (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized with the PrimeScript^® RT reagent kit (TaKaRa, Kusatsu, Japan). All qRT-PCR reactions were performed with a qRT-PCR assay Kit (Vazyme, Nanjing, China) and ABI 7500 real-time detection system (Applied Biosystems, Waltham, MA, USA). Primers used for qRT-PCR analysis are listed in Table S1. All data were normalized to actin gene expression, and relative changes in gene expression levels were analyzed with ABI 7500 SDS software (Applied Biosystems), which automatically set the baseline. Data from three biological replicates were used to calculate the means and standard deviations. The experiment was repeated three times.

Zhang Y., Chen W., Shao W., Tan S., Shi D., Ma H, & Chen C. (2022). FaSmi1 Is Essential for the Vegetative Development, Asexual Reproduction, DON Production and Virulence of Fusarium asiaticum. Journal of Fungi, 8(11), 1189.

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Quantitative Real-Time PCR Analysis

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Total RNA was extracted from the adherent cells and tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using HiScript III 1st Strand cDNA Synthesis Kit for qPCR (cat. no. R312; Vazyme Biotech Co., Ltd.). The thermocycling conditions of the RT were as follows: Remove genomic DNA at 42°C for 2 min; first strand cDNA synthesised at 25°C for 5 min, 37°C for 45 sec and 85°C for 5 sec. qPCR was subsequently performed using the ChamQ Universal SYBR qPCR Master Mix (cat. no. Q711 Vazyme Biotech Co., Ltd.) and the primers provided in Table I on an ABI 7500 Real-Time PCR machine (Applied Biosystems Inc.). The thermocycling conditions of the qPCR were as follows: Denaturation at 95°C for 5 min; 40 cycles at 95°C for 10 sec and 60°C for 30 sec; and a final dissociation stage (95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec) was added at the end of the amplification procedure. The data were analyzed using the ABI 7500 SDS software (Version 2.0.6, Applied Biosystems Inc.). The relative mRNA expression levels were calculated using the 2^−ΔΔCq method (27 (link)) and normalized to the GAPDH reference gene.

Tang J., Meng Q., Shi R, & Xu Y. (2020). PRMT6 serves an oncogenic role in lung adenocarcinoma via regulating p18. Molecular Medicine Reports, 22(4), 3161-3172.

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Isolation and Quantification of Fungal RNA

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To isolate total RNA, the mycelial plugs of each strain were inoculated into liquid YEPD (10 mg/ml peptone, 3 mg/ml yeast extract, 20 mg/ml glucose) and cultured at 25°C for 2 days in a shaker (175 rpm). RNA was isolated from mycelia with the RNeasy kit (Tiangen Biotech. Co., Beijing, China). First‐strand cDNA was synthesized with the PrimeScript^® RT reagent kit (TaKaRa). The real‐time PCR amplifications were conducted in an ABI 7500 detection system (Applied Biosystems) (Zheng et al., 2014). The primers used for Quantitative RT‐PCR assay are showed in Table S1. The expression level of the measured gene in all samples was normalized to actin gene expression, and relative changes in gene expression levels were analysed by ABI 7500 SDS software (Applied Biosystems), which automatically generates the baseline. Data from three biological replicates were applied to calculate the mean standard deviation.

Shao W., Lv C., Zhang Y., Wang J, & Chen C. (2017). Involvement of BcElp4 in vegetative development, various environmental stress response and virulence of Botrytis cinerea. Microbial Biotechnology, 10(4), 886-895.

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Real-Time Fluorescence qPCR Data Analysis

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After completing the real-time fluorescence q-PCR, the obtained data were analyzed by ABI7500 SDS software (Applied Biosystems, Inc.), and the relative expressions of each gene in different samples were calculated by the 2^−ΔΔCt method based on the Ct value of the target gene and the internal reference gene. In this process, ΔΔCt = (CT target gene – CT internal reference) experimental group – (CT target gene-CT internal reference) control group, the data were processed by SAS statistical software. Afterward, the significance test was conducted after logarithmic transformation. The difference shall be considered significant (*) if P < 0.05, the difference would be considered a highly significant difference (**) if P < 0.01.

Fu R., Tang W., Zhang H., Zhang Y., Wang D, & Chen W. (2022). Study on the mechanism of inhibiting patulin production by fengycin. Open Life Sciences, 17(1), 372-379.

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Quantifying Prophenoloxidase Expression in Macrobrachium rosenbergii Hemocytes

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The relative expression of M. rosenbergii prophenoloxidase in the Hemocyte was measured by quantitative real time polymerase chain reaction (qRTPCR). qRT-PCR was carried out using a ABI 7500 Real time Detection System (Applied Biosystems) in 20 μl reaction volume containing 4 μl of cDNA from each tissue, 10 μl of Fast SYBR_ Green Master Mix, 0.5 μl of each primer (20 pmol/ll) (MrProAE-III F1: AACAACTGGGA GTCACCCTTGAGT; MrProAE-III R2: TGACG GCATC TCTGGACAACTTCA) (Arockiaraj et al., 2012 ) and 5 μl dH2O. The qRT-PCR cycle profile was 1 cycle of 95 °C for 10 s, followed by 35 cycles of 95 °C for 5 s, 58 °C for 10 s and 72 °C for 20 s and finally 1 cycle of 95 °C for 15 s, 60 °C for 30s and 95 °C. The same qRT-PCR cycle profile was used for the internal control gene, elongation factor primers were designed based on EST of M. rosenbegrii (Table 1). After the PCR program, data were analyzed with ABI 7500 SDS software (Applied Biosystems). To maintain consistency, the baseline was automatically set by the software. All data were given in terms of relative mRNA expressed as means ± standard deviation. To compare the relative MrProI mRNA expression, statistical analysis was performed using one way ANOVA and mean comparisons were performed by Tukey's Multiple Range Test using SPSS 11.5 at 5% significant level (Fig. 3).

Alinejad T., Bin K.Q., Vejayan J., Othman R.Y, & Bhassu S. (2015). Proteomic analysis of differentially expressed protein in hemocytes of wild giant freshwater prawn Macrobrachium rosenbergii infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV). Meta Gene, 5, 55-67.

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Urinary Exosomal miR-193a Diagnostics

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Statistical analysis was performed by GraphPad Prism version 6 (GraphPad Software Inc., La Jolla, CA, USA). Raw threshold cycles (Ct) values were imported from ABI7500 SDS software (Applied Biosystems, Foster City, CA, USA), and relative expression levels for each miRNA were calculated using the comparative Ct method. Mann–Whitney U-test was used to compare gene expression levels between different groups, and ROC curve was utilized to assess the diagnostic performance of urinary exosomal miR-193a. P < 0.05 was considered statistically significant. All of the results are presented in means ± standard deviation.

Huang Z., Zhang Y., Zhou J, & Zhang Y. (2017). Urinary Exosomal miR-193a Can Be a Potential Biomarker for the Diagnosis of Primary Focal Segmental Glomerulosclerosis in Children. BioMed Research International, 2017, 7298160.

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Quantitative RT-PCR Analysis of Fungal Gene Expression

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RNA samples were isolated with the RNAsimple kit (Tiangen) from germ tubes grown for 24 h in YEPD liquid medium (2% glucose, 0.3% yeast extract and 1% peptone). First-strand cDNA was synthesized with the PrimeScript® RT reagent kit (TaKaRa). All quantitative RT-PCRs were performed with an ABI 7500 real-time detection system (Applied Biosystems, Foster City, CA, USA). The primers used for quantitative RT-PCR analysis are listed in Table S1. The expression of the measured genes in each sample was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression, and relative changes in gene expression levels were analysed with ABI 7500 SDS software (Applied Biosystems), which automatically set the baseline. Data from three biological replicates were used to calculate the mean and standard deviation.

Zheng Z., Hou Y., Cai Y., Zhang Y., Li Y, & Zhou M. (2015). Whole-genome sequencing reveals that mutations in myosin-5 confer resistance to the fungicide phenamacril in Fusarium graminearum. Scientific Reports, 5, 8248.

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Quantification of Apoptosis and Immune Genes

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The TRIzol reagent was used to extract the total RNA from cells (Invitrogen, USA). The first-strand cDNA was synthesized following the usage information (Promega, USA). The expression levels of apoptosis and immune-related genes were presented in an Applied Biosystem 7500 Real-time PCR System, with gene primers listed in Table 1. β-actin was used as an internal standard, and the comparative Ct method with the formula 2 -△△CT was chosen for calculating the relative gene expression levels (Livak and Schmittgen, 2001) . SYBR® Select Master Mix kit was used for the standard qRT-PCR (Life Technologies, USA) on an ABI 7500 SDS software (Applied Biosystems, USA).

Meng X., Li F., Wang X., Liu J., Ji C, & Wu H. (2020). Toxicological effects of graphene on mussel Mytilus galloprovincialis hemocytes after individual and combined exposure with triphenyl phosphate. Marine pollution bulletin, 151.

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Quantitative Real-Time PCR for SMN Genes

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The copy number of two SMN genes, SMN1 and SMN2, was determined by a quantitative real-time PCR assay as previously described (15 (link)). Briefly, 50ng of genomic DNA was amplified in the presence of 300 nM of forward and reverse primers and 100 nM of FAM dye-labeled MGB probes (Applied Biosystems, Carlsbad, CA). The PCR was conducted as described previously (15 (link)), with an exception in using RNase P as the internal endogenous control (Applied Biosystems, Carlsbad, CA). The copy number of SMN genes was determined by using the ABI7500 SDS software and the CopyCaller v2.0 software from Applied Biosystems.

Kariya S., Sampson J.B., Northrop L.E., Luccarelli C.M., Naini A.B., Re D.B., Hirano M, & Mitsumoto H. (2014). Nuclear localization of SMN and FUS is not altered in fibroblasts from patients with sporadic ALS. Amyotrophic lateral sclerosis & frontotemporal degeneration, 15(0), 581-587.

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