Mitsuhashi, M., Tomozawa, S., Endo, K. and Shinagawa, A.
Notes: The authors developed a method to reverse transcribe poly(A)+ RNA from leukocytes without using oligo(dT) immobilized on a 96-well plate. Four RNA targets, as well as a synthetic control RNA, were reverse transcribed using MMLV Reverse Transcriptase (1X reverse transcription buffer [50mM KCl, 10mM Tris-HCl (pH 8.3), 5.5mM MgCl2, 1nL/µL Tween 20], 1.25 mM each dNTP and 4 units of Recombinant RNasin® Ribonuclease Inhibitor), then quantitated by real-time quantitative PCR. The authors varied the concentration of MMLV Reverse Transcriptase, incubation time, and primer/template ratio] to obtain the maximum yield. Small quantities of MMLV Reverse Transcriptase were sufficient to reverse transcribe short synthetic RNA and abundant RNAs, but approximately 100 units was required for other RNAs. The synthetic control RNA was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. (3456)