Hello Research Gate Community,

Wondering if anyone has had issues migrating from the RNAscope Multiplex Fluorescent Assay V1 to the V2 version for mouse brain tissue?

We had the V1 kit working perfectly and were hybridizing 3 different probes easily in both adult and P3 mouse brain tissue. This was using all of the standard conditions outlined in their V1 manual (fresh frozen brain tissue, 15' fixation on slides with cold 4% PFA, 30 minute RT digestion with Protease IV). Great signal, low background, etc.

Sadly when we exhausted the last of our V1 kits and were forced to migrate to V2, everything stopped working. Issue one was that the new pretreatment conditions including the H2O2 step destroyed our postnatal tissues. We fixed this by switching to a 30' RT digestion with Protease III (adult tissues were fine with same time in Protease IV). After we solved this issue, we next found that we were not able to get any signal from either our validated probe set (that worked great with the V1 kit) or the positive control probes. After dropping the post-fix time to 15' with cold 4% PFA, we finally got some signal with the 3-PLEX positive control probes, but only with the UBC, which isn't really a fair target given how highly it's expressed. To make matters worse, when we attempt to develop all 3 channels of the 3-PLEX positive control, we get nothing AND lose the UBC signal.

Quite frustrated at this point as we are close to wrapping something up and need to get this to work. Biotechnique technical support has not been all that helpful and claimed this was and "RNA degradation problem" on our end. Given that we prepared fresh brain samples just for this experiment using the same preservation and storage conditions (snap freeze in OCT, cut 10 uM cryotome sections, slides on dry ice until they are stored at -80C before post-fix, etc.), I can't imagine this is the issue.

If anyone has experienced this and overcome it, or has an optimized protocol for adult and postnatal brain tissue for the V2 kit, I would greatly appreciate your assistance.