Optimization of the Isolation and Amplification of RNA From Formalin-fixed, Paraffin-embedded Tissue: The Armed Forces Institute of Pathology Experience and Literature Review.
Author(s): Krafft AE, Duncan BW, Bijwaard KE, Taubenberger JK, Lichy JH
Publication: Mol Diagn, 1997, Vol. 2, Page 217-230
PubMed ID: 10462613 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of RNA extraction method on RT-PCR success from FFPE liver and lymph node specimens.
Summary of Findings:
The authors report (data not shown) that digestion in 1% SDS and greater than 0.5 mg/mL proteinase K yielded the most consistent amplification of albumin. There was no effect of EDTA concentration (1-20 mM) in the digestion buffer or incubation time (6 versus 16 h) at 55 degrees C. Precipitation with lithium chloride was not successful, but precipitation with isopropanol and glycogen was successful. Guanidium thiocyanate-phenol chloroform allowed for extraction of amplifiable RNA but only when used in conjunction with proteolytic digestion.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Protein digestion Proteinase K (0.125-2 mg/mL)
Analyte Extraction and Purification Cell/tissue permeabilization 0.5% SDS
1% SDS
2% SDS
Analyte Extraction and Purification Incubation duration/condition 6 h
16 h
Analyte Extraction and Purification Analyte isolation method Guanidium thiocyanate-phenol chloroform
EDTA/SDS digestion with isopropanol precipitation
RT-PCR Specific Targeted nucleic acid Albumin