Modification of 4-thiouridine (4sU) disrupts reverse transcription. (A) Overview of the strategy for Roadblock-qPCR. (B) Modification of 4-thiouracil using N-ethylmaleimide (NEM) and iodoacetamide (IAA). (C) NEM and IAA interfere with the reverse transcription of RNA. A 121 nt in vitro transcribed RNA was synthesized with either UTP or 4sUTP. RNAs were treated with EtOH, DMSO, NEM, or IAA (see Materials and Methods) and reverse transcribed using a FAM6-labeled primer with the Protoscript II reverse transcriptase (NEB). cDNA products were then analyzed by PAGE. (D) Length distribution and base conversions in cDNA from NEM-treated RNA. (Top panel) cDNA from NEM-treated RNA prepared in C was analyzed by capillary electrophoresis (fragment analysis). Approximate fragment sizes are shown above. Locations of U nucleotides in the RNA sequence are highlighted as indicated. (Bottom panel) Partial sequences (nucleotides 26–77 of the + strand) of three full-length cDNAs from C. T-to-C base conversions in sequence are highlighted in red.
