RNA was extracted using an RNeasy Plus Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Total RNA (1 μg) from leukemia cells was reverse transcribed with the ReverTra Ace qPCR RT kit (Toyobo Co. Ltd., Osaka, Japan). qPCR using the SYBER Green method was carried out with the Thunderbird SYBER qPCR Mix (Toyobo Co.) on a Thermal Cycler Dice Real-time PCR instrument (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Real-time PCR results were calculated according to the following protocol: Relative expression level=2−ΔCt, where ΔCt=Ct (gene of interest) - Ct (housekeeping gene). The c-MYC primers used for qPCR were: forward, 5′-TTCGGGT AGTGGAAAACCAG-3′ and reverse, 5′-CAGCAGCTCGAA TTTCTTCC-3′. The GAPDH primers used for qPCR were: forward, 5′-GACGCTGGGGCTGGCATTG-3′ and reverse, 5′-GCTGGTGGTCCAGGGGTC-3′ (32 (link)). The cyclin G2 primers used for qPCR were: forward, 5′-ATCGTTTCAAG GCGCACAG-3′ and reverse, 5′-CAACCCCCCTCAGGTA TCG-3′ (33 (link)). The P21/CIP1 primers used for qPCR were: forward, 5′-CGATGCCAACCTCCTCAACGA-3′ and reverse, 5′-TCGCAGACCTCCAGCATCCA-3 (34 (link)).
Thermal cycler dice real time pcr instrument
Manufactured by Takara Bio
Sourced in Japan
The Thermal Cycler Dice Real-time PCR instrument is a laboratory equipment used for amplifying and detecting specific DNA sequences in real-time. It is designed to perform polymerase chain reaction (PCR) analysis.
Automatically generated - may contain errors
2 protocols using thermal cycler dice real time pcr instrument
1
Quantitative Analysis of Gene Expression in Leukemia Cells
2
Quantifying HO-1 Gene Expression in Cancer Cells
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RNA was extracted using an RNeasy Plus Mini kit (Qiagen, Inc.) according to the manufacturer's instructions. Total RNA (1 µg) from the cancer cells was reverse transcribed using the ReverTra Ace qPCR RT kit (Toyobo Co., Ltd.). qPCR using the SYBER-Green method was performed with Thunderbird SYBER qPCR Mix (Toyobo Co.) on a Thermal Cycler Dice Real-time PCR instrument (Takara Bio, Inc.) according to the manufacturer's instructions and as previously described (22) . The heme oxygenase-1 (HO-1) primers used for qPCR were as follows: Forward, 5'-CAG GCA GAG AAT GCT GAG TTC-3' and reverse, 5'-GCT TCA CAT AGC GCT GCA-3' (23) . The GAPDH primers used for qPCR were as follows: Forward, 5'-GAC GCT GGG GCT GGC ATT G-3' and reverse, 5'-GCT GGT GGT CCA GGG GTC-3' (22) . The amplification efficiencies of HO-1 and GAPDH were 0.980 and 0.984, respectively.
Statistical analysis. Values were compared using the two-tailed Student's t-test. Differences among multiple groups was analyzed by one-way ANOVA followed by Tukey's post hoc test, using GraphPad Prism 8 software. P<0.05 was considered to indicate a statistically significant difference.
Statistical analysis. Values were compared using the two-tailed Student's t-test. Differences among multiple groups was analyzed by one-way ANOVA followed by Tukey's post hoc test, using GraphPad Prism 8 software. P<0.05 was considered to indicate a statistically significant difference.
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