RNA Input Recommendations
The protocol is optimized for 25–1000 ng of high-quality total RNA from a human sample. Lower amounts can compromise ligation and lower yield. Determining the input amount for other species and quality levels requires further optimization.
Include a DNase treatment with the RNA isolation method. DNase treatment must be done while running your preferred extraction method. The DNase treatment ensures sample purity and accurate quantification. Before starting the protocol, quantify the total RNA with a fluorometric method intended specifically for RNA. Assess quality using a fragment analysis method.
The following figures provide example traces for Universal Human Reference (UHR) total RNA input.
Example Bioanalyzer Trace
2100 Bioanalyzer System trace using RNA 6000 Pico Kit
Example TapeStation Trace
4200 TapeStation System trace using RNA ScreenTape
