Chimeric padlock probes and HS Library Preparation Kit
At CARTANA, we have a ISS Starter Program. Every new user of the technology is enrolled in this program to get started and successful with the technology. This program provides individual technical support based on your specific tissue samples and experimental design.
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Chimeric padlock probes
CARTANA’s padlock probes are designed using a proprietary algorithm 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, mouse or relevant species). Probes showing cross-reactivity with other RNA transcripts are excluded. Each probe is specific to all published variants for a specific transcript.
We design 4 padlock probes per gene.
When selecting the gene to be targeted, do we need to pay attention to their overall expression levels?
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 padlock probe only bind to mRNA in the cytoplasm or can they also target RNA within the nucleus?
Padlock probes are designed to detect mature RNA (RefSeq annotations are used as references for probe design) and can hybridize to all mature RNA in the cytoplasm and the nuclei.
Probe design and assay conditions are optimized for the detection of mature RNA, and most of them are localized in the cytoplasm. However, detection of pre-mRNA is also possible by targeting intronic sequences.
The anchor sequence is common to all probes and different than the gene-specific ISS barcode.
Below is a sketch of a chimeric padlock probe:
A probe targeting the anchor sequence is used to visualize all ISS spots in the tissue samples, with one general fluorescent label, irrespective of their barcodes (in Cy3, Cy5 or Alexa Fluor® 750 channel, depending on the choice of labeling mix) together with DAPI counterstaining of the nuclei. This is done at the end of the HS Library Preparation protocol.
Imaging the library preparation results prior to the ISS procedure has two purposes:
- To validate the library preparation using the number of ISS spots and the fluorescent intensity of ISS spots. However, the image of the sequencing library does not reveal which transcript an ISS spot represents (this is done during the ISS procedure).
- The image of the HS Library Preparation is the reference to align the sequencing cycles during the ISS image processing steps.
HS Library Preparation Kit
CARTANA currently offers Library Preparation kits that allow the detection of hundreds of genes in one single experiment, with high specificity and single-cell resolution. CARTANA offers small and large 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.
No it is not possible to test several panel on the same tissue section.
How many rolling circle amplification each circle will undergo, basically what is the fluorescent signal?
This depends on the efficiency of the RCA reaction in situ. It is estimated that for a circle of 60-90 bp, the polymerase is able to make approximately 1000 copies/hour for an in vitro reaction (https://doi.org/10.1093/nar/26.22.5073). We use an overnight RCA incubation time so, in principle, this will lead to ISS spots that are estimated to contain 104 copies of each circle.
How long does it take for sequencing 600 genes in 100 cells within a field of view with this new approach?
Since CARTANA ISS is a high throughput method, we do not calculate the time needed to perform ISS based on field of view with limited number of cells but on complete sample area. We consider one sample an area of 1 cm2
The total assay time is independent of the number of genes sequenced.
The HS Library Preparation Kit requires 3 days in total, with 2 overnight incubations:
The ISS Kit requires about 2 hours of hands-on and 1-6 hours per sequencing cycle:
However time for imaging and analysis will vary depending on your imaging system and your data processing and analysis pipeline. , i.e. light source, filter cube exchanging speed, computing capacity etc…
We estimate that each sample (1 cm2) needs approximately one day for image processing and analysis.
CARTANA ISS technology can be combined with cell membrane staining or immunostaining. Staining will be performed after in situ sequencing as the tissue stays intact during the procedure.
The performance of combined ISS/immunostaining 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.
ISS technology is quantitative as one RNA transcript is detected as an ISS spot, therefore it is possible to quantify gene expression levels and compare different experimental conditions.
However, absolute quantification is always challenging, due to some bias in detection (factors like RNA structure, sequence GC content all affect it), but changes in expression levels can be observed and measured using CARTANA ISS. We can also observe different levels of expression between different genes, we estimate that 5-fold or higher variation in expression levels can be quantified.
No, performing the HS Library Preparation and ISS with a lower number of targets, does not reduce either the hands-on, nor the imaging time.
Rolling circle amplification (RCA) is a well-charachterized single molecule amplification reaction (https://doi.org/10.1038/898). Therefore, each ISS spot is the result of one single hybridized and ligated probe.
Additionally, even if the different chimeric padlock probes targeting one transcript would hybridize efficiently and generate different ISS spots, they will be in such close proximity that it will not be possible to resolve individual dots.
We design 4 probes per targeted gene in order to increase efficiency and sensitivity.
We estimate that CARTANA ISS can detect about 10% of all expressed transcripts in cells. Therefore, if your transcript is expressed at 100 copies/cell, we expect to detect about 10 signal dot/positive cell.
The number of ISS spots that can be resolved depends directly on the microscope hardware settings. In-house, we use a 20x objective with 0.75 numerical aperture. ISS spots are between 0.5-1 µm big, so the question comes down to how many ISS spots can be resolved with that optical settings. An average number of a cell of 20 µm of diameter is in the 103 orders of magnitude.
When using 20X magnification, we estimate that we can detect 100-200 dots/ cells. This number increases to 500-1000 dots/cell when using 40X magnification.
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.
For Fresh Frozen tissue samples, we recommend to use 10 µm thickness and for FFPE tissue samples 5 µm thickness ensuring a single layer of cells in the tissue section.
As CARTANA ISS an in situ technique, we sequence the mRNA transcripts directly in the tissues meaning that we can link each sequenced mRNA transcript to a specific cell.
How do you take account the consistency transcript levels from different sections of the same sample?
CARTANA ISS is a reproducible assay. To improve consistency and reproducibility between samples, we highly recommend to prepare and process sections as one batch.
Reproducibility is good between CARTANA ISS experiments, but in order to merge data, some batch corrections might still be required.
- 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/or Alexa Fluor® 750 (749/775)
- Objectives 4X, 20X or 40X.
- Digital camera (pixel size 5-7 µm)