Random priming is widely used in the first strand cDNA synthesis. Random Primer Mix is a ready-to-use optimized mixture of hexamers and anchored-dT primer (dT23VN). A mixture of hexamers and anchored-dT primer provides even and consistent coverage of the RNA template population across a wide range of RNA template concentration. In contrast, traditional random priming by hexamer has poor coverage of 3´ end of RNA templates (1,2,3).
We recommend using Random Primer Mix for reverse transcription of the following RNA templates:
RNA without poly(A) tail such as ribosomal RNAs.
RNA with strong secondary structures.
Partially degraded RNA samples.
Target regions at 5´ end of a long messenger RNA transcript.
Figure 1: Various amounts of human spleen total RNA (2 μg to 20 pg) were reverse transcribed using ProtoScript First Strand cDNA Synthesis Master Mix Kit (NEB #E6300 ) in the presence of 6 μM Random Primer Mix (panel A) or 3.5 μM random hexamers (panel B). About 1/10th of the cDNA products were then analyzed in duplicate qPCR reactions using a pair of primers specific for the beta actin gene with DyNAmo™ FLASH SYBR® Green qPCR Master Mix on a Bio-Rad IQ5® system.
Recommended Usage: Random Primer Mix (60 µM) contains 35 µM hexamers and 25 µM dT23VN and 1 mM dNTPs. We recommend using Random Primer Mix at a final concentration of 6 µM.
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