Sensiscript RT Kit (200)

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Publication protocol

Reverse transcription of RNA: RT was performed using four different reverse transcriptases and appropriate conditions. In all reactions, cDNA was syn-thesized in 20 µl from 2.0 µl of MCF-7 or LNCaP RNA that was diluted from concentrations of 0.1–100 ng/µl in DEPC water. Superscript II RT, MMLV RNase H– (Gibco BRL) or MMLV (GenHunter) were used under conditions described by the RNAimage kit (GenHunter). For each reaction, RT buffer (25 mM Tris–Cl, pH 8.3, 37.2 mM KCl, 1.5 mM MgCl2, 5 mM DTT) and 20 µM dNTP, 200 nM H-T11C anchor primer (5′-AAGC-TTTTTTTTTTTC-3′) were used. Reactions were incubated at 65°C for 5 min, then 100 U RT enzyme was added and incubated at 37°C for 60 min and 75°C for 5 min. Reactions containing 2.0 µl of Omniscript or Sensiscript RT (Qiagen, Santa Clarita, CA), 2.0 µl 10× RT buffer, 2.0 µl 5 mM dNTP (final concentration of 500 µM), 2.0 µl of 10 µM H-T11C (final concentration of 1 µM), 1.0 µl RNase inhibitor (10 U/µl) and 10.0 µl DEPC-treated water were incubated at 37°C for 60 min and then at 93°C for 5 min.

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