FAQ Tissue sample preparation
At CARTANA, we have a ISS Starter Program. Every new user of the technology is enrolled in this program to start successfully with the technology. This program provides individual technical support based on your specific tissue samples and experimental design.
If you have further questions, please contact [email protected]
Is it necessary to use RNA Stabilisation Solution treatment prior to freezing Fresh Frozen samples at -80°C for sample preparation?
We usually recommend working in RNAse free conditions (as much as it is possible) during dissection and sample preparation. If tissue collection is fast and samples are directly frozen or incubated in fixation solution (3.7% PFA or 10% NBF), it is not necessary to use RNA Stabilisation Solution. It is however possible to add a treatment in RNA Stabilisation Solution prior to freezing if you wish.
CARTANA ISS is compatible with all tissue samples from all species. Before moving forward with In Situ Sequencing of more than 100 genes, you can start by using the CARTANA Library Preparation Optimisation Kit that enables you to detect 2 control genes (individually or simultaneously) and optimise the assay conditions specific to your samples (mainly the permeabilisation conditions).
CARTANA ISS technology can also be used to study viral gene expression in tissue samples. Dou D et al. have used ISS Library Preparation technology to label viral genomes.
Dou D et al. Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method. Cell Reports (2017). DOI: 10.1016/j.celrep.2017.06.021
Why do you recommend cutting FFPE sections at 5 µm thickness while you recommend cutting for Fresh Frozen cryosections at 10 µm?
We recommend to cutting fresh frozen samples at 10µm because it is basically the thinnest thickness you can get using a cryostat without too many technical challenges. With thicker sections, the number of cell layers becomes too high and it can become challenging to decode the In Situ Sequencing signals. Regarding FFPE tissue samples, 5 µm is usually the standard thickness used in most laboratories (some even cut at 3 µm). It is challenging to cut thicker FFPE sections without any folding or breaking. Usually >10 µm FFPE sections tend to detach and break during experiments.
Yes, it is possible to perform ISS of TMA. However, as the TMA cores come from different samples that are often handled and prepared differently, it is expected to see variability between cores. As TMA use a surface bigger than 1 cm2, imaging the sequencing cycles will take longer.
To reduce the autofluorescent background in tissue samples such as kidney or lung, it is possible to add autofluorescence quenching steps in the CARTANA HS Library Preparation protocol and at each sequencing cycle of the CARTANA ISS protocol. This protocol has been tested successfully on post-mortem human brain and also on lung samples.
No. Fixation, when performed according to CARTANA’s recommendations, does not affect ISS sensitivity or resolution. However, in the case of FFPE samples, it can be that suboptimal RNA integrity in archival samples affects the overall performance of the assay.
For Fixed Frozen tissue samples, we recommend preparing samples as below:
- Perfuse animal with 3.7% PFA (optional)
- Post-fix samples in 3.7% PFA for 24 hours at 4°C
- Cryo-protect samples with a sucrose gradient
- Embed sample in OCT and store at -80°C (for up to 6 months, for optimal RNA preservation)
For Fixed Frozen tissue samples, we recommend working with 10-14 µm sections. Furthermore, we recommend to work with SuperFrost Slides for all tissue sample preparation methods.
We recommend cutting fresh sections when possible. If storage is needed, we recommend storing sections for up to 4 weeks at -80°C. After this period of time, RNA preservation might be affected.
Using CARTANA ISS, you can use any sample size to perform the experiment as far as it is within a microscope slide format (25×75 mm). This is a hardware limitation since this is defined by the microscope stage or imaging device format. However, please note that we consider one sample as 1 cm2 and you will need to calculate the reagents needed as well as the overall imaging and image processing time according to your sample size.
Fixation conditions, when performed according to CARTANA’s recommendations, do not affect ISS sensitivity or resolution. However, in the case of FFPE samples, suboptimal RNA integrity in archival samples may affect the overall performance of the assay. This is mainly due to variable handling, fixation and storage conditions. For these reasons, we recommend using quality control probes included in the CARTANA HS Library Preparation Kit to validate your samples. Detecting RPLP0 (low expressor) allows you to assess the RNA quality in your samples. Ideally for CARTANA ISS, we would need a RIN value >7 for optimal results. However, we know we can still work on samples with RIN values between 5.5 and 7 but lower performance is expected.