What is the purpose of the different masking levels available in the Stellaris® Probe Designer? Which level should I use?
The Stellaris Probe Designer software lets you design probe sets with adjustable specificity. Please read this article on designing Stellaris RNA FISH probes for step by step instructions on increasing probe count. At levels 0, 1, and 2 the target sequence remains non-masked and oligos are laid down according to the optimal TM and other parameters selected (number of oligos, length, and spacing). At levels 1 and 2, oligos with known difficult sequences or simple repeats such as G-strings, are not allowed. At levels 3 and up, species-specific masking is performed to avoid cross-hybridization to RNAs commonly expressed at high to very high levels.
We advise selecting the most stringent setting possible for your organism, while still generating a full probe count of 24 or more. In the case that you are not able to generate at least 24 probes in your set, you should try decreasing the masking level and trying again. If your species is not listed in the pull down menu, then level 2 is the most stringent available for "other" organisms. The software will not anticipate every scenario and additional bioinformatic analysis is prudent, according to the particulars of the investigation. We advise both careful choice of the target RNA, and BLASTing individual probe sequences against the appropriate database, especially if the intent is to generate a probe set that tolerates or discriminates between similar transcripts.