What requirements should FFPE samples meet for scMLPSeq experiments?

Answer:
To ensure stable nuclei quality and optimal probe hybridization performance, FFPE samples are recommended to meet the following conditions:

• Fresh tissues should be fixed within 2 hours after collection 
• Fixation should be performed using 10% neutral buffered formalin (NBF) for 24–48 hours 
• FFPE blocks are recommended to be stored for no longer than 2 years 
• FFPE blocks should be stored at or below room temperature 
• For FFPE scroll samples, sealed storage at 4°C is recommended, and experiments should ideally be performed within 48 hours 
• Sections placed on glass slides are not recommended (too thin and difficult to remove).
• Biopsy FFPE samples are not recommended (insufficient cell number for capture).

scMLPSeq is fundamentally dependent on RNA target integrity, probe hybridization efficiency and post-fixation nucleic acid accessibility. Over-fixation, prolonged storage, or improper storage conditions may result in: additional RNA degradation, reduced probe binding efficiency, lower ligation-extension efficiency, reduced effective cell recovery and gene detection. 

Samples that do not meet the recommended conditions are not necessarily unusable, but experimental risk may increase substantially. High-risk situations include:

• Delayed fixation after tissue collection 
• Excessively long fixation times 
• Old FFPE blocks stored at room temperature for prolonged periods 
• FFPE scrolls stored under unstable conditions 

For high-risk samples, small-scale pilot testing is strongly recommended before initiating formal projects.