CARTANA’s padlock probes are highly specific and can be used to detect splice variants and also mutations at a single nucleotide resolution.
The Library Preparation Kit can detect up to 600 targets simultaneously.
We can design any probe targeting any gene. We just need to have the exact sequence or accession number of the RNA target to design the probe and manufacture them.
The CARTANA padlock probes consist of a pool of 4 probes per gene.
CARTANA’s padlock probes are designed and tested in silico. CARTANA’s probe design algorithm is validated to select oligonucleotides with compatible design criteria and melting temperature for optimal hybridization and minimal cross-hybridization to off-target sequences. The padlock probes are blasted against the entire transcriptome (human AND mouse). Probes showing cross-reactivity with other RNA transcripts are excluded. Each probe is specific to all published variants for a specific transcript.
We do not reveal the exact sequence of the probe as this is proprietary information. We can share the ~ 50 nucleotide region targeted by each probe if needed.
CARTANA offers small and large HS Library Preparation kits.
We consider one sample as an area of 1 cm2 (if your sample is smaller and you can fit many sections in 1 cm2 , we still consider this as one sample).
The small Library Preparation kit is enough to process 5 samples of 1 cm2 while the large Library Preparation kit contains enough reagents to process 15 samples of 1 cm2.
The CARTANA Library Preparation kit has a shelf life of 1 year (when reagents are stored in the recommended conditions).
The CARTANA probes have a shelf life of 2 years.
CARTANA ISS technology can be combined with IHC. IHC will be performed after In Situ Sequencing as the tissue stays intact during the procedure.
The performance of combined ISS/IHC is however dependent on the epitope and the antibody, indeed, the CARTANA ISS protocol uses several formamide steps that can affect the integrity of proteins. We are currently developing solutions to overcome these challenges.
The CARTANA ISS technology is compatible with Fresh Frozen, Fixed Frozen and FFPE tissue samples.
Yes, the CARTANA ISS sensitivity is the same in all tissue samples preparations. However, in general FFPE samples, and particularly clinical samples, can have sub-optimal RNA integrity that may lead to lower detection efficiency.
CARTANA ISS is compatible with all tissue samples from all species. Before moving forward with the In Situ Sequencing of hundreds of genes, you can start by using the CARTANA Library Preparation Optimization kit that enables you to detect 2 control genes (individually) and optimize the assay conditions specific to your samples (mainly the permeabilization conditions).
Yes, highly expressed genes will cause optical crowding. In the selection of genes, this has to be taken into consideration. We recommend you selecting high expressed genes that are exclusively specific to certain cell types so that they do not overcrowd lower expressed important marker genes. But we also have customized solutions that increase the resolution and that we can discuss with you on case to case basis.
Does the CARTANA ISS technology enables sub-cellular resolution? Is it possible to quantify single molecules inside the cell nuclei?
The CARTANA ISS technology achieves single cell resolution and it is possible to detect pre-mRNA or lncRNA in the nuclei. It is also possible to define sub-cellular localization of specific transcripts.
Can one compare gene expression differences between different sections from a control versus experimental mouse for example?
Yes, as CARTANA ISS is quantitative, it is possible to compare expression levels between different experimental conditions.
Below are the imaging requirements for CARTANA ISS results:
- Epifluorescence microscope with autofocus function.
- Automated (x, y, z) stage with the stage controller.
- Light source and filter cubes for optimal imaging of DAPI (358/461), Alexa Fluor® 488 (495/519), Cy3 (550/570) , Cy5 (650/670) and Alexa Fluor® 750 (749/775)
- Objectives 4X, 20X and 40X.
- Digital camera (pixel size 5-7 µm)
- PC and data storage with specifications suitable for imaging and processing data ranging from 4TB to 60TB.
Currently, we only provide the coordinates. In order to compute genes/cells there are different solutions that we will be happy to recommend. For instance, for cell typing analysis where the knowledge about cell types exists, the prior information can be used in junction to more accurately define cell boundaries: http://insitu.cortexlab.net/ .
For in-house analysis and mapping, we use MATLAB but CARTANA is currently developing a user-friendly platform for data exploration.