Total RNA was isolated with TRIzol reagent (Invitrogen), and all RNAs were reversely transcribed into cDNA using an Exscript RT-PCR kit (TaKaRa) following the manufacturer's instructions. The oligonucleotide primer pairs were as follows: 5'-ACGACCTGACCGCCATTTG-3' (forward) and 5'-CATGGAACGGACAGCGTTTG-3' (reverse) for HDAC4; 5'-CTCATCAGTTGCCACTTCCACATA-3' (forward) and 5'-AGCAATTCATCTGTGCTTTCATGTC-3' (reverse) for HIF-1α. All amplifications and detections were performed using an Applied Biosystems Prism 7900 system (Applied Biosystems, Foster City, CA), an ExScriptSybr green QPCR kit (TaKaRa) and the following program: 1 cycle of 30 sec at 95°C followed by 40 cycles of 5 sec at 95°C and 20 sec at 60°C. Statistical analyses were performed using the 2-△△CT relative quantification method. The assay was repeated three times in triplicate.
Applied biosystems prism 7900 system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems Prism 7900 system is a real-time PCR instrument designed for gene expression analysis. It features a 96-well capacity and can perform quantitative and qualitative nucleic acid analysis.
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Lab products found in correlation
6 protocols using applied biosystems prism 7900 system
1
Quantifying HDAC4 and HIF-1α Gene Expression
2
ERCC1 Expression Quantification Protocol
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For each cell line, total RNA from 3×106 cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNAs were then reverse transcribed into cDNAs for Real-time PCR analysis using an ExScript RT-PCR kit (Takara, Shiga, Japan). Oligonucleotide primers for ERCC1 were 5′-CGTGCTGTACCTCTCGC-3′ (forward primer) and 5′-CTGAGGAACGGTTCCTG-3′ (reverse primer), and primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 5′-GGCCTCCAAGGAGTAAGACC-3′ (forward primer) and 5′-CAAGGGGTCTACATGGCAAC-3′ (reverse primer). Amplification and detection were carried out in the Applied Biosystems Prism 7900 system (Applied Biosystems, Foster City, CA, USA) using an ExScript SYBR Green QPCR kit (Takara) and the following conditions: 95°C for 10 sec; 40 cycles of 95°C for 5 sec, 62°C for 31 sec; followed by a 30-min melting curve analysis to verify the primer dimers. Statistical analysis was performed using the 2−∆∆CT relative quantification method.
3
Quantitative Analysis of PEBP1 Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA was then reverse transcribed into cDNA with the ExScript reverse transcription-polymerase chain reaction (RT-PCR) kit (Takara, Tokyo, Japan). The expression status of PEBP1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined by quantitative PCR. GAPDH was used as the loading control. The following oligonucleotide primer was used: PEBP1 forward, 5′-CTCCGATTATGTGGGCTCGG-3′ and reverse, 5′-GGTGGTCTCCAGATCGGTTG-3′; and GAPDH forward, 5′-CGACCACTTTGTCAAGCTCA-3′ and reverse, 5′-AGGGGAGATTCAGTGTGGTG-3′. All amplifications and detections were carried out using the Applied Biosystems Prism 7900 system (Applied Biosystems, Foster City, CA, USA). The assays were performed in triplicate.
4
Quantitative PCR Analysis of RNA Samples
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Total RNA from 3 × 106 cells for each cell line was isolated by using Trizol reagent (Invitrogen, Carlsbad, CA). All RNAs were then reversely transcribed into cDNAs that were suitable for real-time PCR analysis using the ExScript RT-PCR kit (TaKaRa, Japan). Oligonucleotide primers for RPS7 were 5′-GTCGTCTTTATCGCTCAGAG-3′ (Forward primer) and 5′-TGTCAGAGTACGGCTCCTG-3′ (Reverse primers). Oligonucleotide primers for HIF-1α were 5′-GAACGTCGAAAAGAAAAGTCT-3′ (Forward primer) and 5′-CCTTATCAAGATGCGAACTCA-3′ (Reverse primers). Oligonucleotide primers for GAPDH were 5′-GGCCTCCAAGGAGTAAGACC-3′ (forward primer) and 5′-CAAGGGGTCTACATGGCAAC-3′ (reverse primers). All amplifications and detections were carried out in the Applied Biosystems Prism 7900 system (Applied Biosystems, Foster City, CA) using the ExScript Sybr green QPCR kit (TaKaRa) and the following program: 95°C for 10 s, one cycle; 95°C for 5 s, 62°C for 31 s, 40 cycles; followed by a 30-min melting curve collection to verify the primer dimers. Statistical analysis was performed using the 2−ΔΔCT relative quantification method.
5
Wip1 Gene Expression Quantification
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Total RNA was isolated with Trizol reagent (Invitrogen), and 1 μg RNA was reversely transcribed into cDNA using the Exscript RT-PCR kit (TaKaRa) according to the manufacturer's instructions. Primer pairs for cDNA amplification were as follows: 5′ -GCCAGAACTTCCCAAGGAAAG-3′ (forward) and 5′-GGTTCAGGTGACACCACAAATTC-3′ (reverse) for Wip1; 5′-GGCCTCCAAGGAGTAAGACC-3′ (forward) and 5′-CAAGGGGTCTACATGGCAAC-3′ (reverse) for GA PDH. All amplifications and detections were carried out in the Applied Biosystems Prism 7900 system (Applied Biosystems, Foster City, CA) using the ExScript Sybr green QPCR kit (TaKaRa, Japan) and the following program: 1 cycle of 15min at 95°C followed by 35 cycles of (10 sec at 95°C, 20 sec at 60°C). Statisticalanalyses were performed using the 2−ΔΔCT relative quantification method. The assay was performed three times in triplicate.
6
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNAs of Hey and OVCA433 cells were isolated using Trizol reagent (Invitrogen, Life technologies, USA) and reverse transcribed into cDNA using a PrimeScript™ RT Master Mix (Perfect Real Time; Takara Biotechnology, Shiga, Japan). Real-time PCR was carried out in an Applied Biosystems Prism 7900 system (Applied Biosystems, Life technologies, USA) with TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Biotechnology, Shiga, Japan) using the following conditions: an initial denaturation at 95 °C for 30 s, followed by 95 °C for 5 s, 55 °C for 30 s and 72 °C for 30 s, 40 cycles, after which melting curve analysis was performed to check the specificity of amplification. Each sample was tested in triplicate and Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) was used in parallel reactions as internal control. Three independent experiments were carried out for final analyses using the 2-ΔΔCT relative quantification method. The primer sequences used are listed in Supplementary Table S2.
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