Nucleic acid quantity and quality from paraffin blocks: defining optimal fixation, processing and DNA/RNA extraction...

Area of Science:

• Pathology
• Molecular Biology
• Biochemistry

Background:

• Nucleic acid extraction from formalin-fixed paraffin-embedded (FFPE) tissues is crucial for molecular pathology.
• Standardized protocols for optimizing DNA/RNA quality and quantity are lacking.
• Fixation, processing, and extraction methods significantly impact downstream molecular applications.

Purpose of the Study:

• To evaluate the impact of different tissue fixation methods and durations on DNA and RNA quality.
• To assess the influence of paraffin embedding and processing conditions on nucleic acid yield.
• To determine the optimal nucleic acid extraction techniques for FFPE tissues in molecular diagnostics.

Main Methods:

• Nine tissue samples were subjected to freezing, formalin fixation (<24h, 7 days), or molecular fixative (<24h, 7 days).
• Conventional and rapid processing methods were employed.
• DNA and RNA were extracted using in-house and commercial kits (WaxFree, RecoverAll).
• Extracted nucleic acids were analyzed using PCR and RT-PCR for varying amplicon sizes.

Main Results:

• Molecular fixative (MF) consistently preserved high-quality DNA and RNA, performing comparably to frozen samples across various conditions.
• Formalin fixation was effective only for shorter amplicons and short fixation durations (<24h).
• The WaxFree kit yielded higher DNA success rates but poorer RNA results, while RecoverAll performed suboptimally with prolonged formalin fixation.

Conclusions:

• Molecular fixative, irrespective of fixation length, and rapid tissue processing effectively preserve large DNA and RNA fragments in FFPE tissues.
• These optimized techniques are recommended for improved performance in downstream molecular diagnostic assays.