The Dual-Ribozyme RNA Ladder and RNA-Calibrated ssDNA Marker System for Inexpensive RNA Size Standards

RNA size markers are essential for denaturing Urea-PAGE analysis, but commercial RNA ladders are expensive, available only in fixed size ranges, and require careful handling to prevent RNA degradation. Here, we developed a modular dual-ribozyme platform for preparing RNA size standards from single T7 transcription templates. Each construct contains a central RNA segment flanked by hammerhead and HDV ribozymes, enabling cotranscriptional self-cleavage to generate a predictable set of RNA products. The SYSU25 construct reproducibly produced short RNA marker bands at 25, 50, and 75 nt, whereas the SYSU100 construct generated six products ranging from 100 to 600 nt. The experimentally observed ladder patterns were reproducible using either an in-house or commercial T7 transcription system. A 500 μL in-house transcription preparation of either SYSU25 or SYSU100 provided sufficient material for approximately 50 analytical gel lanes. We further prepared synthetic ssDNA marker sets (S6D and M4D) and calibrated the S6D ladder against RNA standards of known length on 10%, 12%, 15%, and 20% Urea-PAGE gels. This calibration demonstrated that ssDNA and RNA do not migrate solely according to nucleotide length and provided gel-dependent RNA-equivalent size assignments for the S6D bands within the calibrated range. Together, the dual-ribozyme RNA ladders and RNA-calibrated ssDNA markers provide a low-cost reference system for the analysis of short- to medium-length RNA.