Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues.
We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93-100%) than for qPCR (82.4-95%). Correlations between qPCR(FROZEN), the gold standard, and bDNA(FFPE) ranged from 0.60 to 0.94, similar to those from qPCR(FROZEN) and qPCR(FFPE). Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management.
Knudsen
Beatrice S
BS
Fred Hutchinson Cancer Research Center, Division of Public Health Sciences, Seatle, Washington, USA. bknudsen@fhcrc.org
Allen
April N
AN
McLerran
Dale F
DF
Vessella
Robert L
RL
Karademos
Jonathan
J
Davies
Joan E
JE
Maqsodi
Botoul
B
McMaster
Gary K
GK
Kristal
Alan R
AR
D000818:Animals; D018070:Biological Specimen Banks; D021121:Branched DNA Signal Amplification Assay / Q000379:methods*; D005557:Formaldehyde / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction; D011471:Prostatic Neoplasms / Q000235:genetics; D012313:RNA / Q000032:analysis*; D016707:Tissue Fixation
D005557:Formaldehyde; D012313:RNA