Efficient FFPE Sample Preparation and Processing Using the VITA Single-Cell Transcriptome Platform: Tips and Insights
Since its launch in 2023, the VITA single-cell transcriptome solution for formalin-fixed paraffin-embedded (FFPE) samples has helped numerous research teams overcome significant challenges previously associated with single-cell transcriptome studies using FFPE samples.
Drawing from invaluable user feedback, we have continuously refined our protocols to enhance user experience and optimize outcomes. In this article, we summarize key tips and insights for users of the VITA single-cell transcriptome solution for FFPE samples. These essential practical insights and tips aim to streamline FFPE sample preparation and processing on the VITA platform, helping researchers achieve smoother and more efficient workflows, and high-quality single-cell transcriptome data.
I. Tips for FFPE Sample Preparation
Tip 1: Refine FFPE Tissue Roll Thickness.
For optimal results, use FFPE tissue rolls with a thickness between 10 and 20 μm. Rolls thinner than 10 μm can lead to increased nuclear fragmentation, while rolls thicker than 20 μm may hinder reagent penetration due to the density of the tissue.
Tip 2: Avoid Using FFPE Block Edges.
Avoid selecting FFPE rolls from the edges of the block, as prolonged air exposure and reduced tissue content in these areas can compromise sample quality and integrity.
Tip 3: Adjust Sample Volume.
Ensure that the tissue volume is small enough to be fully immersed in the reaction solution (as shown in the figure below). Insufficient submersion may result in incomplete reagent interaction and, consequently, incomplete sample processing.
II. Tips during FFPE Tissue Deparaffinization and Rehydration
Tip 1: Minimize Tissue Loss During Deparaffinization
Exercise caution when aspirating the deparaffinizing solution during wash steps to avoid inadvertently aspirating tissue fragments. For samples with fragmented tissue (example shown in the figure below), a brief centrifugation can be used to pellet the tissue, reducing the risk of sample loss during subsequent washes.
Tip 2: Ensure Complete Tissue Spreading After Ethanol Rehydration.
Following the ethanol rehydration process, confirm that the tissue is fully spread (as shown in the figure below) to maximize reagent interaction and enhance reaction efficiency. If curling persists, carefully use a pipette tip to unfold the tissue, ensuring optimal surface contact with the reagents.
III. Tips for FFPE Tissue Dissociation
Tip 1: Eliminate Small Debris Post Tissue Dissociation.
If small debris remains following tissue dissociation (example shown in the figure below), adjust the centrifugation speed to target and remove lighter particles. While this approach effectively eliminates smaller fragments, it may also result in the loss of some intact nuclei, so exercise caution when working with low-cell-count samples.
Tip 2: Utilize Flow Cytometry for Impurity Removal When Needed.
If the nuclear suspension following tissue dissociation contains significant impurities that are difficult to remove by centrifugation (example shown in the figure below), using flow cytometry to sort and purify the nuclei can be an effective alternative.
By implementing these preparation and processing strategies, researchers can significantly improve both the efficiency and the reliability of FFPE sample workflows on the VITA platform.
We will be sharing additional tips on VITA single-cell transcriptome experiments to help improve your experimental efficiency and data quality. Stay tuned for more updates!
