Table 1.

Troubleshooting table.

Step	Problem	Possible reason/cause	Solution/suggestions
4.5	Low concentration of RNA in the homogenate	• The pellet is not completely resuspended	• Resuspend again by pipetting up and down and incubate on ice for 10–15 min
5.5	Low polyribosome peaks are observed	• Degradation of RNA • “Runoff” of ribosomes	• Increase the quantity/concentration of RNAse inhibitors • Keep the temperature low (0–4); Keep all the solutions and materials cold and RNAse free • Tumors must be cooled/frozen as quickly as possible after harvesting and stored at –80; Keep all solutions cold and work on ice
5.5	Nothing is detected in the polyribosome profiles	• Polyribosome extraction didn’t work properly • Poor quality of the sample • Not enough tumor tissue material was used • Sensitivity is too low	• Increase the number of strokes during the mechanical lysis (add one or 2 more strokes) • Tumors must be cooled/frozen as quickly as possible after harvested and stored at –80 • For optimal polyribosomal profiles results, it is recommended to load a 10–20 OD260 • Increase the sensitivity of the UA-6 absorbance monitor by changing the setting to a higher sensitivity
5.5	Poor quality profiles	• Disrupted gradients	• The gradients should be handled with caution in order to minimize any perturbation