Our Technology- In Situ Sequencing

Learn how you can apply our technology in your research.

In Situ Sequencing (ISS) is an innovative method of nucleic acid analysis performed directly in situ on the tissue section, preserving the spatial information.

Our newly developed New Generation ISS Technology allows researchers to analyse either fresh/fixed frozen or FFPE samples and rapidly create single cell gene expression maps of up to 600 genes.  The technology ensures high performance (high specificity and high throughput) and reliable reproducible data.

“Single cell transcriptomics has enabled molecular understanding of the fundamental building block of life now its time to take the next step and understand how these building blocks act together in tissues and organs, which is the functional unit of animals.

Let’s go from molecular cell biology to molecular tissue biology!”

-Professor Mats Nilsson, P.h.D, Science For Life Laboratory, Stockholm University

Next Generation In Situ Sequencing Technology

CARTANA’s new ISS technology contains chimeric padlock probes that enable highly specific in situ library preparation of up to 600 different RNA sequences without the need for complementary DNA (cDNA) synthesis by reverse transcription (RT).

Library Preparation

RNAs of preserved tissue are fixed in their original location inside the cell by a short fixation step

After that, the highly specific chimeric padlock probes in your CARTANA High Sensitivity Library Preparation KIT will hybridize to the RNA corresponding to your genes of interest.

The closed circle is then copied locally by a DNA polymerase in a rolling circle amplification (RCA) reaction. This generates an ISS spot of the detected RNA transcript that includes multiple copies of the ISS barcode sequence corresponding to the detected gene at the original position of the RNA target.

HS Library Prep Kit Technical Note
Chimeric Padlock Probe
Rolling Circle Amplification
Sequencing cycles & Decoding

Then CARTANA ISS Kit chemistry is used to decode barcodes from target-specific ISS spots generated during the Library Preparation with the CARTANA HS Library Preparation Kit.

The closed circle is then copied locally by a DNA polymerase in a rolling circle amplification (RCA) reaction. This generates an ISS spot of the detected RNA transcript that includes multiple copies of the ISS barcode sequence corresponding to the detected gene at the original position of the RNA target.

  • Decoding starts with the hybridization of the 1st adapter probe (AP) pool to the ISS spots and binding of the sequencing pool (SP) probes to the adapter probes (AP). A sequencing pool (SP) containing 4 different fluorescent labels that bind to the anchor and barcode sequences from the ISS spots.
  • After imaging, the sequencing signal is chemically removed to perform the next sequencing cycle. In total, 6 sequencing cycles are required for full decoding of up to 600 targets.
Technical workflow of In Situ Sequencing
  • Adapter probe pool hybridization leads to a target-specific ISS spot displaying a unique fluorescent signal in each cycle.
  • Once all 6 sequencing cycles are analyzed, each ISS spot provides a unique fluorescent barcode identifying the targeted RNA identified with a gene marker.
In Situ Sequencing Cycle
ISS Kit Technical Note
Tissue Map

The ISS technology results into a map of gene expression of up to 600 selected genes on the nature morphology of the tissue.

spatial genes expression of fresh frozen human breast cancer sample

In Situ Sequencing of 31 transcripts in Fresh Frozen human breast cancer samples using CARTANA ISS technology.