Figure 1.

Comparison of 5′-RLM-RACE and MBRACE methods. The initial steps are the same in both methods, in which an RNA linker (striped bar) is attached to siRNA-cleaved mRNA (solid line). After cDNA synthesis by reverse transcription, a first round PCR reaction is set up using one primer specific for the linker sequence and a second primer specific to the gene target and product generated (dotted line). For standard 5′RACE (left), a second round PCR is performed using internal primers (Nested). Products are then analyzed by agarose gel electrophoresis, and amplicons of the predicted size are excised, cloned and sequenced to confirm that the product is derived from siRNA-mediated cleavage. For MBRACE, the first round PCR products are also used as template for a second round with the same nested primers, but the addition of a molecular beacon spanning the junction between the linker and cleaved mRNA enables amplification to be detected in real-time using a LightCycler® 480.