Total RNA was extracted from P29mtP29 and P29mtB82M cells using TRI reagent (Sigma-Aldrich), and 1 µg of total RNA was reverse-transcribed with ReverTraAce qPCR RT Master Mix (TOYOBO, Osaka, Japan). Quantitative real-time PCR was performed with THUNDERBIRD SYBR qPCR Mix (TOYOBO) in a total volume of 20 µl in a Thermal Cycler Dice Real Time System TP860 (TaKaRa, Shiga, Japan): 95 °C for 1 min followed by 40 cycles of denaturation (95 °C for 15 sec) and extension (60 °C for 1 min). Experiments were performed in triplicate. After amplification, dissociation curve analyses were performed to confirm the amplicon specificity for each PCR run. The relative levels of gene expression in P29mtP29 and P29mtB82M cells were normalized against mouse Gapdh. Quantification was performed using the 2−ΔΔCT method. The primer sets are shown in Supplementary Table S6 .
Thermal cycler dice real time system tp860
Manufactured by Takara Bio
Sourced in Japan
The Thermal Cycler Dice Real Time System TP860 is a compact and versatile real-time PCR instrument. It is designed for efficient and accurate DNA amplification and detection, featuring a compact design and advanced temperature control technology.
Automatically generated - may contain errors
4 protocols using thermal cycler dice real time system tp860
1
Quantifying Gene Expression in Cell Lines
2
Quantitative Real-Time PCR Protocol
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Quantitative RT-PCR (qRT-PCR) was performed on cDNA using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) and 0.3 μM primers in a 20 μl volume. The reactions were run on a Thermal Cycler Dice Real Time System TP860 (TaKaRa, Shiga, Japan). The PCR protocol consisted of an initial denaturation step at 95 °C for 1 min and 40 cycles of denaturation (95 °C for 15 s) and extension (60 °C for 1 min). Dissociation curve analyses were performed to confirm the PCR product identity and to differentiate specific amplification from non-specific products by denaturation (95 °C for 15 s), annealing (60 °C for 30 s), and slow heating to 95 °C. The mRNA expression level of each gene was normalized to GAPDH. The specific primer sets are shown in Supplemental Table SI .
3
Quantitative Real-Time PCR Analysis
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Total RNA isolation and reverse transcription were performed as described above. Quantitative real-time PCR was done using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) in a total volume of 20 μl on a Thermal Cycler Dice Real Time System TP860 (TaKaRa, Shiga, Japan): 95 °C for 1 min and 40 cycles of denaturation (95 °C for 15 s) and extension (60 °C for 1 min). Experiments were performed in triplicates. Following amplification, dissociation curve analyses were performed to confirm the amplicon specificity for each PCR run. The relative level of gene expression in human and mouse cells was normalized against human GAPDH or mouse Gapdh, respectively. Quantification was performed using the 2−ΔΔCT method. The specific primer sets are shown in Supplementary Table 3 .
4
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA extraction with TRIzol reagent (Sigma-Aldrich) and semiquantitative RT-PCR were performed as described previously.46 (link) Real-time PCR was performed with cDNA using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) and 0.3 μM primers in a total volume of 20 μl. The reactions were run on a Thermal Cycler Dice Real Time System TP860 (TaKaRa, Shiga, Japan). The PCR protocol consisted of an initial denaturation step at 95 °C for 1 min and 40 cycles of denaturation (95 °C for 15 s) and extension (60 °C for 1 min). Dissociation curve analyses were performed to confirm the PCR product identity and to differentiate specific amplification from non-specific products by denaturation (95 °C for 15 s), annealing (60 °C for 30 s) and slow heating to 95 °C. The results were evaluated as relative expression levels standardised using the expression level of GAPDH mRNA. The forward and reverse primers used for the PCR reactions are listed in Supplementary Table II .
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