Nucleic Acids Res. 1993 Jul 25;21(15):3597-8

Hirzmann J, Luo D, Hahnen J, Hobom G.

Determination of messenger RNA 5'-ends by reverse transcription of the cap structure.

The mRNA cap can template an extra C in the first strand cDNA. “50% of the cDNAs of capped mRNA molecules had an extra G”

“poly(A)+ RNA was obtained using Hybond-mAP paper (Amersham). 1 µg of poly(A)+ -RNA in 9.65 µL of water was heated to 60°C for 3 min, cooled on ice, added to 4 µL of 5 × RT buffer (1 × RT buffer is 40 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 40 mM KCl, 2 mM DTE), 3 µL dNTPs (10 mM each), 0.25 µL (10 units) of RNasin (Promega), 2.5 µL of XhoI-(dT)17 oligonucleotide primer (300 pmol, 0.5 µg/µL). and 0.6 µL (16 units) of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer). The reaction was continued at 42 °C for 2 hours, and excess XhoI-(dT)17 primer and substrates were removed by glass powder purification followed by ethanol precipitation.”