Table 1.

Troubleshooting

Problem	Reason	Solution
Smeary gel	RNA is not completely degraded after reverse transcription (see Fig. 5).	Use pH paper to make sure the pH is 13–14 after NaOH addition (step 25).
	Sodium acetate from the final precipitation step is present in the ssDNA sample.	Wash pellet with 70% ethanol (v/v) before resuspending in gel loading dye (step 28).
Low signal in lanes with DMS	Insufficient reaction of DMS with RNA	Increase DMS concentration and/or incubation time (steps 5 & 6).
Weak full-length bands and strong product bands in lanes with DMS	Over-reaction of DMS with RNA	Decrease DMS concentration and/or reaction time (steps 5 & 6).
Extensive banding in the absence of DMS treatment; good extension in sequencing lanes	Poor extension by RT caused by problems with DMS treatment step	Make sure that phenol is not being carried over into the primer extension reaction. If necessary, perform a chloroform extraction after step 11. If footprinting an RNP, check whether bands are present in the ‘no DMS’ lanes, both with and without protein. If the bands are only present with the protein, the protein preparation is likely contaminated with RNase activity. Use an RNase inhibitor or purify the protein further to remove the contaminating RNase.
Extensive banding in the absence of DMS treatment and in sequencing lanes	RNA stock is degraded	Check by end-labeling the RNA and analyzing the length distribution of the prep by PAGE.
	Poor extension by RT caused by reaction conditions	RT (step 18) is sensitive to Mg2+ concentration. Make sure the final Mg2+ concentration is 3–4 mM in excess of dNTPs (which bind stoichiometrically to one Mg2+ ion). Try increasing the temperature of the primer extension reaction (up to 50 °C), as the RNA may form secondary structure that inhibits RT, and higher temperature may destabilize this structure.
	Poor extension by RT caused by inactive enzyme	Replace RT with new enzyme stock (step 18).
	Poor choice of RT enzyme	Switch to a different RT (step 18). While traditionally primer extension is performed using AMV-RT, engineered versions of MMLV such as Superscript (Invitrogen) are also used and may be more effective for a given RNA.
	Primer binds in more than one position on the RNA. (If using more than one primer, this problem will be apparent because the problem will be primer -specific.)	Construct a new primer (see Primer Extension). Make sure that there are not additional complementary regions within the RNA.
	Additional bands preset because primer is heterogeneous at its 5′-end	Purify the primer carefully by PAGE (see “32P-labeling of DNA primers” in Reagent Setup).
No extension in experimental lanes; good extension in sequencing lanes	RNA lost in precipitation after DMS treatment.	Add carrier (like RNA or linear acrylamide) for precipitation (step 14) or increase RNA concentration in footprinting reactions.
	RNA did not resuspend after the pellet was dried	We recommend air drying rather than using a Speedvac (step 14) to prevent this problem.
	Strong inhibition of RT from phenol carryover	Remove the aqueous phase carefully (step 11) to ensure that phenol is not carried over. If necessary, perform a chloroform extraction to ensure complete removal of phenol.
Bands absent or very faint in all lanes, including the band representing the primer	Radioactive material was lost in final precipitation	Check pellet and supernatant with a Geiger counter. If necessary, incubate at −20 °C for 1 hr after adding ethanol (step 29).
	Insufficient label was used in primer extension	Make sure solution of primer is at least 50k dpm/µl.