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

Author(s): Turashvili G, Yang W, McKinney S, Kalloger S, Gale N, Ng Y, Chow K, Bell L, Lorette J, Carrier M, Luk M, Aparicio S, Huntsman D, Yip S

Publication: Exp Mol Pathol, 2012, Vol. 92, Page 33-43

PubMed ID: 21963600 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of preservation method, fixation duration, long-term storage of paraffin blocks, and DNA extraction method on the integrity and amplifiability of DNA and morphological preservation from non-cancerous colon, myometrium, and liver tissue and archival cancerous tissue. Snap-frozen specimens were stored at -80 degrees C. Three 10 um sections were used for DNA extraction.

   Summary of Findings:

   Fixation with Sakura molecular fixative resulted in moderate to severe red blood cell swelling and superimposed eosinophilia which were not present in formalin-fixed tissue. There was no statistically significant difference between DNA extraction success rates with different extraction methods. For all tissue types, the WaxFree DNA kit produced the highest yields, followed by the in-house method, the QIAamp FFPE Tissue kit, and lastly the RecoverAll kit, although lower yields were obtained from colon tissue than from myometrium or liver. Spectrophotometry revealed that although higher yields were obtained using the WaxFree DNA kit, the DNA was of lower purity than that obtained by other methods. Significantly less DNA was extracted from tissue fixed with molecular fixative than from tissue fixed with formalin. PCR success rates differed based on fragment length (smaller fragments were more successful), preservation method (snap-frozen and molecule fixative-fixed specimens had higher success than formalin-fixed specimens), time in fixative (24 h was superior to 7 days for formalin fixation), and DNA extraction method (WaxFree DNA extraction was superior while the performance of other extraction methods depended on preservation method). Only the 268 bp amplicon was amplified from >15 year old archival FFPE blocks. Further, this was only possible when extraction was performed using QIAamp DNA FFPE Tissue kit, WaxFree DNA kit, or the in-house method and not the RecoverAll kit.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Liver
   • Tissue - Uterus

   Preservative Types

   • Formalin
   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified
   • Neoplastic

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Spectrophotometry
   Morphology	H-and-E microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Sakura molecular fixative Snap frozen
   Biospecimen Preservation	Time in fixative	<24 h 7 d
   Storage	Storage duration	<1 y >15 y
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DNA FFPE Tissue kit WaxFree DNA kit RecoverAll kit Phenol-chloroform-isoamyl alcohol extraction
   PCR Specific	Targeted nucleic acid	Beta-globin GAPDH
   PCR Specific	Length of gene fragment	268 bp 536 bp 700 bp 988 bp 1327 bp
   Biospecimen Acquisition	Biospecimen location	Colon Myometrium Liver

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2. Study Purpose

   The purpose of this study was to determine the effects of preservation method, fixation duration, long-term storage of paraffin blocks, and RNA extraction method on the integrity and amplifiability RNA from non-cancerous colon, myometrium, and liver tissue and archival cancerous tissue. Snap-frozen specimens were stored at -80 degrees C. Three 10 um sections were used for extraction.

   Summary of Findings:

   There was no statistically significant difference between RNA extraction success rates with different extraction methods. For all tissue types, the WaxFree RNA kit and in-house TRIzol methods produced the highest yields, followed by the RNeasy and RecoverAll kits. Colon tissue yielded the least amount of RNA. Spectrophotometry reveled that although higher yields were obtained using the WaxFree RNA kit and TRIzol, the RNA was of lower purity than that obtained by the RNeasy and RecoverAll kits. The highest RNA yields were obtained from specimens preserved by snap-freezing or formalin fixation for <24 h. RT-PCR success rates differed based on fragment length (smaller fragments were more successful), preservation method (generally snap-frozen and molecule fixative-fixed specimens had higher success than formalin-fixed specimens for all extraction methods except WaxFree RNA kit), time in fixative (24 h was superior to 7 days for formalin fixation), and RNA extraction method (performance of each was dependent on preservation method). Neither RT-PCR amplicon was amplified from >15 year old archival FFPE blocks, regardless of extraction method.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Liver
   • Tissue - Uterus

   Preservative Types

   • Formalin
   • Frozen
   • Other Preservative

   Diagnoses:

   • Not specified
   • Neoplastic

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Sakura molecular fixative Snap frozen
   Biospecimen Preservation	Time in fixative	<24 h 7 d
   Storage	Storage duration	<1 y >15 y
   Analyte Extraction and Purification	Analyte isolation method	RecoverAll kit RNeasy FFPE kit WaxFree RNA kit TRIzol and chloroform extraction
   RT-PCR Specific	Targeted nucleic acid	Beta-actin
   RT-PCR Specific	Length of gene fragment	621 bp 816 bp
   Biospecimen Acquisition	Biospecimen location	Colon Myometrium Liver

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