Table 2.

Troubleshooting guide for F30-2xdBroccoli imaging in mammalian cells

Problem	Possible reason	Solution
No RNA pellet observed after centrifugation (Alternate protocol 1, step 19)	RNA degraded following contamination with RNase Too little cells were taken for RNA isolation	Ensure RNase-free environment. Use only RNase-free solutions and plasticware Make sure that cells are confluent in the well and did not dye after transfection. Use larger well to grow and transfect cells.
Total RNA concentration is too low	RNA degraded following contamination with RNase	See above
No fluorescent bands observed after DFHBI staining	RNA degraded following contamination with RNase F30-2xdBroccoli does not fold when fused to the RNA in study Transfection failure Too low F30-2xdBroccoli expression level Too short staining time with DFHBI Low concentration of DFHBI in the staining buffer or buffer was used for too many times	See above Test if dBroccoli folds and fluoresces when fused to the RNA in study in vitro. Optimize dBroccoli insertion point if needed. Co-transfect a red fluorescent protein to confirm it. Ensure that the cells are healthy and in a low passage number. Thaw fresh cell batch if needed. Verify the plasmids purity. Re-elute the plasmids if needed. Promoter is too weak. Use stronger promoter, such as U6 or 5S High rate of RNA-F30-2xdBroccoli turnover in cells or undesirable cleavage of the transcript. Inspect your transcript’s sequence for cryptic transcription termination sites or potential destabilizing elements. Insert F30-2xdBroccoli into a different site within your RNA. Extend DFHBI staining time Re-make the staining buffer
No distinct individual bands observed after SYBR Gold staining, smear seen in lanes	RNA degraded following contamination with RNase	See above
No fluorescent population observed when using a flow cytometer	Poor RNA-F30- 2xdBroccoli folding in cells Incorrect DFHBI concentration Cell incubation time with DFHBI is too short	Optimize F30-2xdBroccoli insertion point. Verify that the cells are incubated with 40 μM of DFHBI or DFHBI-1T Extend cell incubation time with DFHBI (DFHBI-1T)
Cell did not attach to the glass-bottom dish	Poor cell health Insufficient glass surface coating	Ensure that cells are healthy and free of contamination. Change the media when it turns yellow. Do not transfect cells when their confluence is low. Ensure that the glass surface is properly coated with poly-lysine and laminin. Increase laminin coating time if needed.
Cells die during the imaging	Media pH change Phototoxicity of the excitation light	Use either an environmental chamber with a controlled level of CO2 or buffer the imaging media with HEPES to maintain pH of 7.4 Do not expose cells to high intensity light for an extended period of time. Aim to expose cells to light only for brief time periods when quickly scanning the fields and then during actual images taking.
No green fluorescence signal in cells under the microscope	Low expression level of RNA-F30-2xdBroccoli in cells Photobleaching Improper concentration of DFHBI	Even when a fluorescent band is seen on a gel after DFHBI staining and a positive population is observed during the flow cytometry analysis, the dBroccoli brightness level may still not be enough for successful fluorescence detection using fluorescence microscopy. Increase expression level by utilization of the strongest promoters, such as U6 or 5S, and ensure no degradation or cleavage of the transcript. Photobleaching is more noticeable when imaging dBroccoli in mammalian cells due to the low expression level of the transcript. Leaving the cells in the dark to allow new fluorophore to bind to Broccoli is critical to restore the signal prior to acquiring an image. Check stock solutions and remake imaging media