What is the multiplexing level (#genes)?
HuluFISH probes can be used to multiplex 7 genes simultaneously at the moment, using a combination of three base dyes (Atto488, Atto565, and Atto647N). This has been validated in the embryonic mouse brain. Theoretically, as one increases the number of dyes, the limit can reach up to 127 genes, but we don't offer this right now.
How sensitive is the method (smFISH-like or padlock-like)?
The sensitivity is smFISH-like. The probe-selection algorithm allows a high sensitivity for even low-expressing genes. In any case, it is way more sensitive than the padlock-like method.
How many probes per transcript do you design for?
We design 32 probes for a gene with adequate length. For a gene smaller than 500 bp, we design as many as possible according to the sequence. Until now we have tested a minimum of 7 probes per gene to work.
What is the difference between HuluFISH and HuluFISH plus probe?
HuluFISH+ is designed is optimally designed for FFPE (Formalin-Fixed-Paraffin-Embedded) samples. Due to the nature of the FFPE sample with higher fluorescence background and lower RNA quality than fresh sample, the smFISH probe for FFPE is required to have a higher intensity to overcome the limitations. HuluFISH plus probe is having 9x more fluorophores than the HuluFISH probe. The super brightness of HuluFISH plus is also allowing imaging of the sample with a regular epifluorescence microscope.
What is the number of fluorophores and in what ratios per probe? For example, for a single fluorophore, do you put on three dyes per probe or just one? For two fluorophores, do you use a total of three dyes, so two of one color and one of the other color?
You are right about that. For a single fluorophore, we put three dyes per probe. For two fluorophores, we use a total of three dyes, two of one color and one of the other color. For your genes it will be as follows:
Gene A: 3R (3 Atto647N)
Gene B: 2Y1R (2 Atto565, 1 Atto647N)
How do you design probes for high expressing genes? Is there a limit to the number of spots per cell that you can detect due to the crowding problem?
For highly expressed genes, we try to assign them into a single channel to avoid crowding in all channels. The limitation is determined by the cell size. The community has estimated about 30000 RNA dots per cell per channel as a general limit.
Can I perform Fiber-FISH on combed single DNA molecules over APTES coated glass slides?
We have a similar experiment running, which is called HuluFISH on Chip. We are using our probe to detect a 500 bp dsDNA on a glass surface. We would be interested to know more about your DNA target size, sequence complexity, and so on. Could you send us more information on that? We can then better answer how likely it is for HuluFISH probes to work in that case.
Is this product used for RNA in situ hybridization assay, such as the RNAScope kit?
Yes, we are very similar to the RNAscope or Stellaris probe if they used any of them. our probe is designed to detect genes with high multiplexity.
Can it be used to pull down specific RNA species from cell lysate to identify RNA-biding proteins?
Yes, we could have TEG-biotin labeled HuluFISH probes for the pulldown of Ribonucleoprotein complexes.
How well does it work on tissue samples?
The probes have been tested on Drosophila embryos, and mouse embryonic brains and spinal cord, Human liver tissue, Human breast tissue, platynereis embryo, etc.