Effects of formalin, methacarn, and fineFIX fixatives on RNA preservation.

Author(s): Dotti I, Bonin S, Basili G, Nardon E, Balani A, Siracusano S, Zanconati F, Palmisano S, De Manzini N, Stanta G

Publication: Diagn Mol Pathol, 2010, Vol. 19, Page 112-22

PubMed ID: 20502189 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the amount and integrity of RNA extracted from fresh frozen tissue with that from tissues fixed in formalin, methacarn, or FineFIX. FineFIX-fixed specimens were preserved and processed using a microwave processor, while formalin and methacarn-fixed specimens were processed conventionally.

   Summary of Findings:

   Total RNA amount recovered was on average 50% lower in formalin-fixed tissues than in tissues fixed in methacarn or FineFIX or fresh frozen tissues. rRNA was degraded in all fixed tissues with RNA integrity number (RIN) values ranging from 2.3 to 2.6 compared with an RIN of 7.4 for fresh frozen tissue where RIN values >6 were defined as good quality RNA. mRNA expression levels, as detected by real time qRT-PCR, were preserved in methacarn-fixed specimens, but lower in formalin and FineFIX-fixed specimens when compared to fresh frozen controls. However, when RT-PCR was used to assess mRNA integrity, specimens fixed in FineFIX showed successful amplification of 438 bp amplicons, similar to methacarn-fixed specimens, whereas only amplicons up to 200 bp long were amplified from formalin-fixed tissue.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Kidney
   • Tissue - Lung
   • Tissue - Prostate
   • Tissue - Gall Bladder
   • Tissue - Breast

   Preservative Types

   • Formalin
   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Electrophoresis
   RNA	Northern blot
   RNA	Real-time qRT-PCR
   RNA	RT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Embedding medium	Conventional paraffin embedding Microwave-assisted paraffin embedding
   Analyte Extraction and Purification	Analyte isolation method	TRIzol reagent Proteinase K-based method
   RT-PCR Specific	Targeted nucleic acid	ACTB
   RT-PCR Specific	Length of gene fragment	77 bp 177 bp 291 bp 438 bp 651 bp
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACTB ATP5B HPRT
   Biospecimen Preservation	Type of fixation/preservation	FineFIX Formalin (buffered) Methacarn Snap frozen

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