Abi 7500 fast instrument

Manufactured by Takara Bio

The ABI 7500 Fast instrument is a real-time PCR system designed for high-throughput genetic analysis. It is capable of performing fast thermal cycling and can accurately quantify nucleic acid targets.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (2 mentions) M mlv transcriptase, by Takara Bio (2 mentions) Abi 7500 fast instrument, by Thermo Fisher Scientific (2 mentions) Sybr premix ex taq 2, by Takara Bio (2 mentions) β actin antibody, by Santa Cruz Biotechnology (1 mentions) Sybr green pcr master mix, by Takara Bio (1 mentions) Primerquest, by Integrated DNA Technologies (1 mentions) Qiazol kit, by Qiagen (1 mentions) Sybr green kit, by Takara Bio (1 mentions)

4 protocols using abi 7500 fast instrument

Effect of HBVDNAPTP1 on CIP4 Expression

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To detect the effect of HBVDNAPTP1 on CIP4 mRNA, total RNA was extracted from THP-1 cells transiently transfected by pcDNA3.1 (-)/myc-His A and pcDNA3.1 (-)/myc-His A-HBVDNAPTP1 using Trizol reagent (Invitrogen) according to the manufacturer's instructions and was used for real time Real time RT-PCR using the ABI 7500 Fast instrument and SYBR Green PCR master mix (Takara). The following specific primers (sense 5′-ACGCTCAATTGAACCCTGC, antisense 5′-ACGATGGTAGAAGGCACAGC) were used to amplify the CIP4 cDNA (Product size: 201bp). G3PDH specific primers (sense 5′- TGTGTCCG-TCGTGGATCTGA, antisense 5′-TTGCTGTTG-AAGTCGCAGGAG) was used as internal reference (Product size: 150bp). Cycling parameters were 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. After PCR amplification, amelting curve was plotted to measure PCR specificity. Real-time PCR results were analyzed using the ΔΔCt method. 2–ΔΔCt represented the average relative amount of mRNA (5 (link)).
To detect the effect of HBVDNAPTP1 on CIP4 protein, total soluble proteins were extracted from the transfected THP-1 cells and separated on 12.5% SDS-PAGE for immunoblotting assay. The expression of CIP4 was probed by monoclonal antibody, and β-actin antibody (Santa Cruz) was used as internal reference. The immunoreactive bands were visualized in an UVP Biospectrum Imaging System.

LUN Y., XU C., CHI Q., WANG X., SUI W, & JIANG S. (2014). CDC42-Interacting Protein 4 Gene Is Down Trans-Regulated by HBV DNA polymerase Trans Activated Protein 1. Iranian Journal of Public Health, 43(3), 282-290.

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Tumor-Derived RNA Quantification by qRT-PCR

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RNA samples extracted from four individual tumours were used as biological replicates for qRT-PCR analyses. Each biological replicate had three technical replicates. For each sample, reverse transcription was performed on 2 μg total RNA by 200U M-MLV transcriptase (Takara). The reaction was carried out at 70 °C for 10 min, 42 °C for 60 min and 70 °C for 15 min. Resulting cDNA was diluted to 800 μl with sterile water. qPCR was carried out in triplicate using an ABI 7500 Fast instrument (Applied Biosystems/Life Technologies). Gene-specific primers were designed using PrimerQuest (Integrated DNA Technologies). The actin gene (Accession no.: ADK11998) was used as an endogenous control. PCR was carried out in 20 μl volume containing 2 μl cDNA, 250 nM forward primer, 250 nM reverse primer, and 1 × SYBR Premix Ex Taq II (TaKaRa) using the following conditions: 95 °C for 3 min, 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. Melting curve analyses were performed to verify the specificity by ABI 7500 Fast instrument Manage software. Relative expression levels were calculated using the 2–^ΔΔCt method (Schefe et al, 2006 (link)). A two group t-test was used for statistical analysis.

Diaz-Montero C.M., Mao F.J., Barnard J., Parker Y., Zamanian-Daryoush M., Pink J.J., Finke J.H., Rini B.I, & Lindner D.J. (2016). MEK inhibition abrogates sunitinib resistance in a renal cell carcinoma patient-derived xenograft model. British Journal of Cancer, 115(8), 920-928.

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Quantitative RT-PCR Analysis of Gene Expression

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For each tissue type, the RNA samples extracted from three individuals were used as biological replicates for qRT-PCR analyses. Each biological replicate had three technical replicates. For each sample, reverse transcription was performed on 2 µg total RNA by 200 U M-MLV Transcriptase (Takara) in a 20 µl volume. The reaction was carried out at 70°C for 10 minutes, 42°C for 60 minutes and 70°C for 15 minutes. The resulting cDNA was diluted to 800 µl with sterile water. qPCR was carried out in triplicate reactions using an ABI 7500 Fast instrument (Applied Biosystems). Gene-specific primers were designed using PrimerQuest (http://www.idtdna.com/Primerquest/Home/Index). The primers used in this study are listed in S1 Table. The Actin gene (Accession no.: ADK11998) was chosen as an endogenous control. PCR was carried out in a 20 µl volume containing 2 µl diluted cDNA, 250 nM forward primer, 250 nM reverse primer, and 1×SYBR Premix Ex Taq II (TaKaRa) using the following conditions: 95°C for 3 min and 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 15 sec. Melting curve analyses were performed to verify the specificity by ABI 7500 Fast instrument Manage software. The relative expression levels were calculated using the 2–^ΔΔCt method [31] (link).

Chen H., Wu B., Nelson D.R., Wu K, & Liu C. (2014). Computational Identification and Systematic Classification of Novel Cytochrome P450 Genes in Salvia miltiorrhiza. PLoS ONE, 9(12), e115149.

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Quantifying Total RNAs in GBM

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Total RNAs in GBM tumor tissues and cells were isolated using Qiazol kit (Qiagen) and TRIzol reagent (Invitrogen). The synthesis of cDNA was performed using GlodScript one-step qRT-PCR Kit (Applied Biosystems). Real-time uorescence quantitative PCR reaction was carried out on ABI 7500 Fast Instrument with SYBR Green Kit (Takara). Primers were dsigned with Primer Express 3.0 software.
Relative quantities were normalized to U6 or GAPDH, and the data were explicated by 2 -ΔΔCt method.

Pan H., Xu J., Wang G., Wang Z., Yao Z., & Zheng Z. (2020). LncRNA SBF2-AS1 Promotes Malignant Phenotypes and Radioresistance of Glioblastoma Cells by Impairing miR-338-3p-targeted MAP4K3 Inhibition.

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