Table 1.

Troubleshooting table

Step	Problem	Possible reason	Solution
52	Slight change in the colour of the NuPAGE LDS Sample Buffer	Low pH of the sample caused by the remaining TCA	Add 1 μl of 1 M Tris buffer pH 7.8
53	Uneven migration of the samples in the gel	Low pH of the sample caused by the remaining TCA	Carefully observe the colour of the samples from Step 52. If the problem persists add a further wash with ice-cold acetone.
55	Additional bands on the autoradiography film - radioactive signal in the negative control at the level of tagged AGO	Cross-contamination of the samples	Repeat the experiment taking care not to mix the samples
	Additional bands on the autoradiography film – at different levels than tagged AGO	Background proteins bound to the beads	Extend the wash in Step 26.
	Radioactive smear in the whole lanes of the gel, visible at autoradiography film	Unspecific RNA bound to AGO-RNA complexes	Extend the wash in Step 26; add more RNace-IT
	Low signal on the autoradiography film	Low expression of tagged-AGO	Optimize conditions of tagged-AGO expression; expose the gel longer
		Low binding efficiency to IgG-Dynabeads	Check binding efficiency by western blot (see comments to Step 25 in the “Anticipated results” section); coat the Dynabeads once more and wash them well directly before the experiment.
		Inefficient cross-linking	Optimize cross-linking conditions
		RNA degradation	Ensure that you work in RNase-free conditions
57	High radioactive signal on the nitrocellulose membrane.	Inefficient Proteinase K treatment or other unrecognized reason	May try adding more Proteinase K, and incubating samples for an additional hour. However, from our experience, RNAs retention on the membrane is a result of preceding steps and cannot be reversed at this stage.
59	Significant radioactivity in the organic phase	Inefficient Proteinase K treatment; remains of protein bind to RNA prevent it from entering aqueous phase	Take fresh Proteinase K aliquot; add more Proteinase K and/or incubate samples longer. See also troubleshooting comments to Step 57.
61	Pellet loose or floating in supernatant	traces of organic phase aspirated during PCI extraction	Take more care collecting upper aqueous phase.
63	RNA pellets difficult to resuspend	Pellets over dried	Take more care drying pellets
75	Samples diffuse from wells while loading	Samples contain leftover ethanol from Step 74	Incubate remaining samples for a few min at 50°C, add more loading dye
76	No PCR products despite strong signal on the autoradiography film	Problems with enzymatic reactions	Replace enzymes, check buffers
		Problems with ligation of sequencing adapters	After resuspending in water, divide the adapters into small aliquots; use fresh aliquot for each experiment; (see comments to Step 76 in the “Anticipated results” section)
	Unique PCR product of approximate size of 120 bp	Only PCR amplified linker dimers visible on the gel; problems with enzymatic reactions	See other troubleshooting comments to Step 76
	PCR products smaller then 140bp migrate in one relatively sharp band	Excessive RNase digestion in Step 17	Optimize RNase digestion conditions. See “Experimental design: RNase digestion” section
79	No bacterial colonies on the plates	Unsuccessful TOPO-TA cloning	Check if your polymerase leaves 3′ A-overhangs in the PCR products; if not add A-overhangs postamplification according to the protocol supplied with the TOPO-TA cloning kit
		Top10 bacteria do not survive transformation	During transformation be very gentle; do not pipette bacteria and do not shake them after heat shock before spreading on the plate
81	Sequencing returns multiple similar sequences that cannot be mapped to the database	RNA contamination coming from commercial enzymes (frequently bacterial rRNA)	Change enzyme supplier
		Problem with 3′ end adapter ligation	miRCat-33 primer used for reverse transcription is complementary to bacterial rRNA. Therefore, a problem with 3′ end adapter ligation results in enrichment of cDNA/PCR product in bacterial rRNA.
	No reads mapping to miRNA in the sequencing result	Significant experimental problems	Repeat the experiment