Table 1.

Troubleshooting

Step	Problem	Possible Reason	Solutions
7, 8, 13	MB does not fluoresce in the presence of the target oligonucleotide	The MB stem-loop structure is too stable The MB loop sequence is too short or too AT rich	The stem should be weakened by decreasing its length and/or GC content The loop length and/or GC content of the MB should be increased
8, 13	MB fluoresces in the presence of a mismatch oligonucleotide	The hybridization of the MB with the mismatch oligonucleotide is more energetically favorable than the formation of the stem-loop MB structure despite the energy penalties of the mismatches	The stem should be strengthened by increase its GC content and/or length
11	MB fluorescence cannot be detected in GFP+ cells	The GFP fluorescence is detectable at other wavelengths, and it is obscuring beacon signals because of its intensity	Instead of Cy3 or FAM dyes, switch to a dye with an even more spectrally distinct emission wavelength such as Cy5 Ensure that your analysis method is using an appropriate excitation laser that avoids exciting GFP and maximizes the light absorbed by the MB dye
15	MB fluorescence cannot initially be detected in positive wells	The MBs have not bound to their targets in the solution	Allow more time for the beacon and target solutions to mix, or mix the solutions using a shake function on the microplate reader, or mix the solutions more vigorously while pipetting
21	Fewer than 80% of the cells are positive for delivery MB signal	Cell membranes are not porous enough for a long enough period of time for MBs to enter the cell	Continue to test other electroporation parameters to increase the transfection efficiency. If transfection cannot be improved, switch to a more easily manipulated cell type
26	The negative control cells are generating a false positive signal with MB targeted towards the gene of interest	The control cells actually do express the gene of interest (verify by RT-PCR) The gene is not expressed but a non-specific interaction in the control cells is causing MB to fluoresce (verify by RT-PCR) The gene is not expressed but a non-specific interaction in the control cells is causing MB to fluoresce in the cytoplasm (verify by RT-PCR and microscopy)	Use a different cell type as a control Check the cells using a microscope to observe subcellular localization. If MBs are apparent in the nucleus, try reducing the time between electroporation and analysis Change the mRNA target region to avoid non-specific interactions. This will require the development of a new MB
26	The positive control cells do not give a strong MB signal	The MB targeting sequence may be inaccessible	Redesign MB to target a different region of the gene to have better accessibility
39	Fewer than 80% of the differentiated cells are positive for delivery MB signal	Cell membranes are not porous enough for a long enough period of time for MBs to enter the cell	Continue to test other electroporation parameters to increase the transfection efficiency. If transfection cannot be improved, normalize all values to the transfection percentage. Since the dyes on the delivery control and cell- specific beacon are spectrally distinct they may be delivered concurrently for easier analysis.
41	The positive control cells do not give a strong cell specific MB signal	The MB targeting sequence may be inaccessible	Redesign MB to target a different region of the gene to have better accessibility