Evaluation of formalin-free tissue fixation for RNA and microRNA studies.

Author(s): Arzt L, Kothmaier H, Quehenberger F, Halbwedl I, Wagner K, Maierhofer T, Popper HH

Publication: Exp Mol Pathol, 2011, Vol. 91, Page 490-5

PubMed ID: 21641900 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 fixative type on the yield, purity and quality of RNA from 10 paraffin-embedded lung cancer specimens. From each specimen, 4 adjacent tissue samples were obtained and fixed in formalin, HOPE, FineFix, or RCL-2 and paraffin-embedded. Blocks were stored at 4 degrees C until sectioning. RNA was extracted from 3-10 4 um sections shown by H%E staining to be composed of 85% tumor cells.

   Summary of Findings:

   While RNA yield was comparable between the 4 fixative types, the purity of the RNA was lower from FFPE specimens than RCL-2-, HOPE- or FineFix-fixed specimens (p<0.001). Further, while the average RIN was 2.1-2.5 for all fixatives, fragments of 2000 bp were obtained from HOPE-fixed specimens, but fragments from RCL-2-fixed specimens had a maximum size of 500 bp, and those from formalin and FineFix-fixed specimens were generally <200 bp. The yield of miRNA was higher from FineFix-fixed specimens than HOPE- (p=0.032) or RCL-2-fixed specimens (p=0.0005), and HOPE-fixed specimens had a higher miRNA yield than RCL-2-fixed specimens (p=0.005). Fixative type affected normalized real-time qPCR measurements of miR-106a (p=0.00003) and miR-155 (p=0.003), and pairwise comparisons showed more miR106a in FineFix-fixed than HOPE-fixed specimens.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin
   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	FineFIX Formalin (buffered) HOPE RCL2
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-106a miR-155

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2. Study Purpose

   The purpose of this study was to determine the effects of specimen storage, extraction method, proteinase K digestion duration, and additional purification on RNA yield and purity from two FFPE lung carcinoma specimens. The two FFPE lung tumor specimens were stored for 1 and 13 years. Proteinase K digestion durations of 15 min, 3 h and overnight were performed in conjunction with RNeasy extraction, and digestions lasting 3 h, 5 h, and overnight were performed in conjunction with RecoverAll extraction.

   Summary of Findings:

   The specimen stored for 1 year yielded at least twice as much RNA as the specimen stored for 13 years. The RNA yield from both specimens increased when the duration of proteinase K digestion increased, but maximum yield was observed after 3 h and 5 h with the RNeasy and RecoverAll kits, respectively. The RNeasy kit yielded more RNA and did a better job at removing the chemical contaminants than the RecoverAll kit. Additional purification with the Turbo DNA-free kit did not improve the RNA purity and reduced yield, regardless of initial purification kit.

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1 year 13 years
   Analyte Extraction and Purification	Protein digestion	15 min 3 h 5 h Overnight
   Analyte Extraction and Purification	Analyte isolation method	RecoverAll nucleic acid isolation kit RNeasy FFPE
   Analyte Extraction and Purification	Analyte purification	With Turbo DNA-free kit No additional purification

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