. Author manuscript; available in PMC: 2008 Oct 3.

Published in final edited form as: Nat Protoc. 2008;3(10):1537–1549. doi: 10.1038/nprot.2008.145

Table 3.

TROUBLESHOOTING

PROBLEM	POSSIBLE REASONS	SOLUTION

ASO does not derepress reporter.	ASO forms strong secondary structure.	Redesign flanking sequences.
	ASO transfection was not efficient.	Co-transfect plasmid and ASO. If derepression is now observed, ASO transfection was inefficient. Repeat ASO transfection or optimize transfection.
	Endogenous miRNA is not abundant enough to repress the reporter.	Check miRNA level by northern blot. Try co- transfection with a “frayed” (guide nucleotide p1 mismatched) siRNA corresponding to the miRNA strand¹⁰⁴ to be sure it is not a problem with the reporter. A pri- miRNA expression vector could also be used.
	Incorrect reporter target site.	If the reporter cannot be repressed, check the miRNA target site in reporter sequence.
	Cholesterol modified ASO was not mixed.	Vortex ASO stock before use and mix ASO-S2 cell mix by tapping the tube.
Cells die after ASO transfection.	Too much transfection reagent or ASO used.	Possible if cell death is seen in miRNA-specific ASO and control ASO transfected samples. Repeat with correct amount of transfection reagent or ASO or replace media after 12-24 h.
	Cell density was too low.	Repeat with correct cell density.
	miRNA inhibited is required for cell survival.	Possible if miRNA ASO kills cells, but control ASO does not. Determine mechanism of cell death. Perhaps the miRNA is anti-apoptotic. Gain of function studies may also be informative (e.g. does overexpression protect cells from apoptosis?).