. Author manuscript; available in PMC: 2017 Apr 19.

Published in final edited form as: Curr Protoc Mol Biol. 2015 Jan 5;109:26.3.1–26.330. doi: 10.1002/0471142727.mb2603s109

Table 26.3.1. Troubleshooting Guide for C. elegans RNAi Studies.

Problem	Possible cause	Solution
RNAi by microinjection
dsRNA solution does not flow from the needle	Needle is closed	Etch or break the needles after pulling to open
	Needle is clogged	Break the tip of the needle to restore flow
		Use a new needle
		Centrifuge dsRNA solution prior to use
Worms do not stick to agarose injection pad	Agarose is not dry enough	Bake agarose pads at least 1 hr at 50°C
No RNAi phenotype observed	dsRNA is degraded	Prepare fresh dsRNA and analyze on 1% agarose gel to check quality (unit 2.5a)
	dsRNA is degraded	Perform positive control for RNAi by injection
	Target gene is refractory to RNAi	Try another region of the gene
	Target gene is refractory to RNAi	Attempt to achieve RNAi by feeding or soaking
RNAi by feeding
No RNAi phenotype observed	dsRNA production is not induced	Test the plates by performing a positive control for RNAi
	dsRNA production is not induced	Prepare fresh NGM/Amp/IPTG plates
	RNAi food is contaminated	Prepare fresh RNAi food. Be sure to include both ampicillin and tetracycline in the starter culture to prevent contamination with another bacterial species. Perform all food preparation carefully.
RNAi by soaking
No RNAi phenotype observed	dsRNA is degraded	Prepare fresh dsRNA and soaking solution