Gene expression analysis using long-term preserved formalin-fixed and paraffin-embedded tissue of non-small cell lung cancer

Abstract

Abstract. This study was performed to evaluate RNA extraction and gene expression analysis of non-small cell lung cancer (NSCLC) using formalin-fixed and paraffin-embedded (FFPE) specimens stored for more than 20 years by quantitative PCR (qPCR) and DNA microarrays. Long-term preserved FFPE materials enable large retrospective studies correlating molecular features with therapeutic response and clinical outcome. qPCR was used to evaluate RNA extraction methods and to compare DNA microarray gene expression profiles of FFPE and fresh frozen (FF) tissue. The Ambion RecoverAll kit appeared to be suited for RNA extraction of long-term preserved FFPE tissues. Microarray analysis using the Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues and identified known NSCLC signature genes in both specimens. We conclude that high quality gene expression signatures can be recognized using the Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality.

Transcriptomic Profiling of Human Peritumoral Neocortex Tissues Revealed Genes Possibly Involved in Tumor-Induced Epilepsy

“…Some of the dysregulated genes could serve as good candidate for biomarkers or targets for seizure management. Our analyses also validate the notion that meaningful genomic data can be acquired from a widely available and valuable clinical resource [15]–[27].…”

Testing an Aflatoxin B1 Gene Signature in Rat Archival Tissues

“…7, 48 qPCR has been used to obtain gene expression data from FFPE samples from different tissue types and organs, often with tumor specimens to improve prognostic accuracy in clinical care. 7, 12, 15, 49 For example, RNA from FFPE samples of 153 breast cancer patients were used to develop an 8-gene qPCR gene expression score of prognostic value that could be reasonably implemented in clinical settings without the need for fresh frozen RNA or more costly commercial signature arrays. 7 One recent study identified a 14 gene expression signature using qPCR with FFPE RNA from a 361 patient cohort to predict survival in nonsquamous, non-small-cell lung cancer.…”

“…4, 11, 12 Although less comprehensive than microarray platforms, quantitative PCR (qPCR) is considered one of the most sensitive methods for comparing a wide range of gene expression levels, it is frequently used to confirm signatures generated by microarray platforms and it is reliable even when the RNA is of low amount and quality. 13 qPCR of RNA extracted from clinical FFPE samples is being used to detect the presence of gene signatures in an effort to improve patient diagnosis and to predict therapeutic response.…”

“…13 qPCR of RNA extracted from clinical FFPE samples is being used to detect the presence of gene signatures in an effort to improve patient diagnosis and to predict therapeutic response. 12, 14, 15 Similarly, our goal was to explore use of qPCR for predictive gene signatures in FFPE tissue blocks stored in a large institutional archive. 16 …”

Semi-Nested Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Successful Quantitation of Cytokeratin mRNA Expression Levels for the Subtyping of Non-Small-Cell Lung Carcinoma Using Paraffin-Embedded and Microdissected Lung Biopsy Specimens

“…FFPE tissue specimens are thought to be unsuitable for genetic examination after long periods of time have elapsed. However, molecular biological technologies for examining archival FFPE samples are now more developed [2, 9, 10, 12, 14, 29, 32, 34]. The possibility of measuring mRNA levels in FFPE tissue specimens has been reported, despite RNA fragmentation [6, 9, 10, 27, 29, 31, 34].…”