Optimisation of DNA and RNA extraction from archival formalin-fixed tissue.

Author(s): Coombs NJ, Gough AC, Primrose JN

Publication: Nucleic Acids Res, 1999, Vol. 27, Page e12

PubMed ID: 10454649 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to optimize DNA and RNA extraction from formalin-fixed paraffin-embedded (FFPE) archival specimens for PCR analysis by comparing 10 extraction techniques with distinct deparaffinization and purification methods.

Conclusion of Paper

The authors identified deparaffinization via a thermocycler coupled with Chelex-100 purification as the optimal DNA extraction method, with 60% of specimens yielding successful PCR results; although specimens extracted with the commercial QIAamp DNA mini kit yielded a comparable PCR success rate of 60%. RNA extracted by the thermocylcer deparaffinization and Chelex-100 purification method resulted in successful amplification of 84% of specimens. Successful PCR analysis of both DNA and RNA extracted from FFPE archival specimens was negatively correlated to the length of paraffin block storage, with significant reductions in PCR efficiency observed in specimens stored for 10 years or longer.

Studies

Study Purpose

The purpose of this study was to compare PCR success rates (for the cytochrome P450 2D6 gene) for DNA isolated from archival FFPE specimens by three different deparaffinization and three different purification techniques with the QIAamp DNA mini kit.

Summary of Findings:

Of the sections deparaffinized with xylene and ethanol, only those that underwent phenol/chloroform purification yielded successful PCR results. All three purification methods resulted in successful PCR amplification of microwave or thermocycler deparaffinization methods. Deparaffinization in a thermocycler followed by Chelex-100 purification was the most efficient DNA extraction method, yielding a PCR product for 61% of specimens, compared to a 60% success rate using the commercial QIAamp DNA mini kit.

Biospecimens

Tissue - Colorectal

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
DNA	PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Deparaffinization	Xylene/ ethanol Microwave oven heating Thermal cycler heating QIAamp DNA mini kit
Analyte Extraction and Purification	Analyte isolation method	Phenol/ chloroform Simple boiling in Tris-EDTA Chelex-100 QIAamp DNA mini kit

Study Purpose

The purpose of this study was to determine if the duration of paraffin block storage affects successful PCR and RT-PCR analysis of DNA and RNA extracted from FFPE specimens by the thermocycler deparaffinization/ Chelex-100 purification method, respectively.

Summary of Findings:

The duration of paraffin block storage at room temperature adversely affected PCR analysis of DNA extracted by thermocycler deparaffinization and Chelex-100 purification. PCR success rates decreased from 65% for specimens stored for less than 5 years to 52% for those stored for 10 years or longer. RNA extracted by an identical method resulted in successful RT-PCR amplification of a beta-actin cDNA fragment in 84% of specimens, and displayed detrimental effects of paraffin block storage similar to those observed for DNA.

Biospecimens

Tissue - Colorectal

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
RNA	RT-PCR
DNA	PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Storage	Storage duration	1-4 yr 5-10 yr 11-20 yr 21-30 yr

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