Selection of Reference genes for normalization of qRT-PCR data derived from FFPE breast tumors.

Author(s): Drury S, Anderson H, Dowsett M

Publication: Diagn Mol Pathol, 2009, Vol. 18, Page 103-7

PubMed ID: 19430294 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine appropriate reference genes for qRT-PCR analysis of FFPE and frozen breast tumor tissue.

   Summary of Findings:

   Importantly, there was a 2800-fold difference in the level of detection of candidate reference genes from FFPE and frozen specimens, the former being lower, using qRT-PCR. After candidate reference gene expression values were obtained using 10 specimens for which both FFPE tissue and frozen tissue was available, the expression stability of each gene was determined using geNorm software. MRPL19, TFRC, and TBP were the most stably expressed genes in FFPE tissue while TBP, PUM1, and KIAA0674 were the most stably expressed genes in frozen tissue. The geometric mean of each of these groups was chosen as the normalization factor for the corresponding preservation method.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACTB GAPDH GUSB KIAA0674 MRPL19 PDGB PUM1 RPLPO TBP TFRC

   View more

2. Study Purpose

   The purpose of this study was to validate the chosen reference genes and corresponding normalization factors for each method of preservation.

   Summary of Findings:

   ERalpha expression correlates (r=0.90) between 24 matched FFPE and frozen tissue samples when chosen sets of reference genes were used for normalization. Furthermore, enzyme immunoassay results correlated with normalized ERalpha mRNA expression data for both FFPE (r=0.87) and frozen tissue (r=0.70) samples. A positive correlation was also seen between 20 matched FFPE and frozen tissue samples when HER2 expression was normalized using the chosen sets of reference genes (r=0.81), and the expression data corresponded to HercepTest findings.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Protein	Immunoassay
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	ERalpha HER2
   Immunoassay Specific	Targeted peptide/protein	ERalpha
   Immunohistochemistry Specific	Targeted peptide/protein	HER2

   View more