Table 5.

Troubleshooting.

Problem	Possible Reason	Solution
Low titer.	Transfection reagents do not have sufficient transfection efficiency.	Test transfection reagents for their efficiencies. Requantify plasmid concentration used for transfection.
	pH of HEK293T cell medium is not optimal for transfection.	Remove HEK293T dishes from incubator immediately before transfection.
	The sequence of the ITRs (inverted terminal repeats) within the plasmid is not intact, thus replication and packaging of the rAAV vector is inefficient.	Check for the presence of SmaI and/or Eam1105I restriction sites within the palindromic ITR sequence. In case of unexpected restriction fragment band pattern, repeat cloning using an intact rAAV vector.
The injection bleb in the subretinal space is not visible under the surgical/stereomicroscope.	The eye shows damages or opacity of the cornea.	Make sure to choose only mice with clear, intact eyes for subretinal injection. Check the eyes under the stereomicroscope.
	Virus suspensions diluted with double-distilled water to adjust the particle concentration do not show optical refraction as clear as the virus suspension in 0.014% Tween/PBS-MK.	Dilute with 0.014% Tween/PBS-MK to adjust virus particle concentration. If the suspension is too viscous, dilute with double-distilled water at a maximum ratio of 1:2 (water-to-virus suspension).
	The injection angle is not optimal.	Adjust the injection angle to ~60°.
Loss of many OS.	Removal of the pigment epithelium and ciliary body after retina isolation with foreceps may result in loss of many OS.	Proceed with the next step without any removal of undesirable tissue.
Protein expression level is too low, thus leading to a too weak or absent fluorescence signal.	Virus suspension is not (completely) injected into the subretinal space.	Make sure to see a clear injection bleb as this indicates the correct injection to the subretinal space. An injection into the upper layers of the eye such as sclera or choroidea causes a bleb outside at the globe. If the needle is intravitreal, it can be clearly and sharply seen under the stereomicroscope. By contrast, a needle in the subretinal space (injection angle at ~60°) appears rather blurred. Keep the injection needle for at least 20 s in the bleb to ensure proper delivery of the desired volume. If possible, perform OCT measurements on the anesthetized mouse after injection to confirm detachment of the retina at the injection position.
	Virus titer has not been determined correctly.	Double check the virus titer. Otherwise, repeat the virus production.