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Comprehensive analysis of fundamental RNA quality factors in archival tissue
Author(s) -
Chung JoonYong,
Williams Reginald,
Guerrero Natalie,
Lim Langston,
Tuttle Kimberly,
Takikita Mikiko,
Luo Yuling,
Hewitt Stephen M.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1147
Subject(s) - rna , rna extraction , fixative , nucleic acid , dna , biology , microbiology and biotechnology , gene expression , gene , lysis , genetics , cytoplasm
Formalin fixed, paraffin embedded (FFPE) tissue is the most common specimen available for application of diagnostic assays on tissue after microscopic examination. However this approach is not molecular biology friendly, with significant damage to both nucleic acids and proteins by degradation and cross‐linking. Extraction of sufficient, quality RNA from FFPE tissue is a barrier to the development of new diagnostic assays. We have optimized a reliable RNA extraction method based on deparaffinization at high temperature coupled with 3‐day lysis at 65°C. The average total RNA yield is 4.5 to 5.5 pg per 1 μm 3 of archival FFPE tissue, and 260/280 ratios are between 1.80 and 1.95. With a new optimized RNA isolation protocol, we are investigated the fundamental factors which influence on RNA integrity within FFPE tissue. Using mouse kidneys as a standardized tissue, we profiled the pattern of RNA degradation based on fixation time, tissue processing time and fixative buffer. Quality was measured by bioanalyzer tracings, RT‐PCR and branched DNA assays of specific genes. Fixation time and tissue processing time have significant impact on RNA quality. Our data suggested that the middle region of gene could be a promising target for RNA analysis. Overall, these date of this study provide fundamental knowledge in studies of gene expression analysis using archival FFPE tissues.