Stem-loop primers were used for reverse transcription of mature miRNAs (Chen et al., 2005).The sequences of Stem-loop and gene-specific primers are summarized in Table 2 . For cDNA synthesis, about 1μg of RNA was mixed with 1 pmol of specific stem-loop RT primers and incubated at 75°C for 5 min. A mix containing 100 units of M-MuLVRT-enzyme (Vivantis, Malaysia), 1x RT-buffer, and 400 μMdNTP were added to the tube. Finally, RT reaction mixtures were incubated at 25°Cfor 15 min, 37°C for 15 min, 42°C for 45 min, and 10 min at 75°C, in a thermal cycler instrument (Eppendorf, Germany). The resulting cDNAs were stored at -20°C prior to RT-qPCR analyses.
Thermal cycler instrument
Manufactured by Eppendorf
Sourced in Germany
The Thermal Cycler Instrument is a laboratory device used for the amplification of DNA samples through the polymerase chain reaction (PCR) process. The instrument precisely controls the temperature and duration of the thermal cycling steps required for DNA replication.
Automatically generated - may contain errors
2 protocols using thermal cycler instrument
1
Stem-Loop RT-qPCR for miRNA Detection
2
Methylation-Specific PCR Analysis of PTEN Promoter
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Genomic DNA (1 μg) of micro-dissected tumor tissues was extracted by the MagMA FFPE DNA Isolation Kit (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. Bisulfite modification was performed by EpiTect Fast Bisulfite Conversion Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. MSP was performed using 100 ng of bisulfite-modified DNA, primers set and Taq DNA Polymerase Master Mix (Ampliqon, Copenhagen, Denmark) in a final volume of 25 μl in thermal cycler instrument (Eppendorf, Hamburg, Germany). MSP were analytically validated using standard methylated DNA as positive control (Chemicon, Temecula, USA) and primary keratinocyte DNA as negative controls. The amplification products were electrophoresed on a 2.5 % agarose gel, stained in sybr green, and visualized by UV transilluminator (Uvitec, Cambridge, UK). The MSP results were reported as methylated samples (a band 173 bp) and unmethylated samples (a band 155 bp). MSP results of PTEN promoter were also confirmed by bisulfite sequencing.
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