Table 5 Troubleshooting

Cells are not viable after seeding onto Terasaki plates

Cells were not viable prior to seeding

Check viability using Trypan Blue dye exclusion or fluorescent stain system (e.g., Calcein AM/Ethidium Homodimer-1) to ensure cells are viable prior to seeding

Terasaki plates are contaminated

Confirm sterility of plates and treat as necessary by UV sterilization and/or alcohol soaking.

Too many/too few cells per well

Inaccurate cell dilution

Confirm cell counting method accuracy and recalibrate any automated instrumentation

Cells are adhering to the sidewalls of the wells

First confirm cells are deposited into the well by visually identifying sidewall-adhered cells. Next, allow cells to settle without disruption after seeding or, alternatively, centrifuge plates for 1minute at 900 rpm to draw cells to the bottom of the well.

No amplification of target gene

Improperly designed primers

Confirm primer design by bulk cell RT-qPCR followed by band sequencing and redesign primers as necessary.

Loss of RNA during harvesting

Reduce number of fluid transfer steps and/or use low-binding tubes and tips.

RNA degradation

Use RNase-free reagents and perform all steps following cell harvesting on ice.