Beatrice S. Knudsen, April N. Allen, Dale F. McLerran, Robert L

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Presentation transcript:

Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin- Fixed Tissues Beatrice S. Knudsen, April N. Allen, Dale F. McLerran, Robert L. Vessella, Jonathan Karademos, Joan E. Davies, Botoul Maqsodi, Gary K. McMaster, Alan R. Kristal The Journal of Molecular Diagnostics Volume 10, Issue 2, Pages 169-176 (March 2008) DOI: 10.2353/jmoldx.2008.070127 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 LOQ of the bDNA assay in archival tissue blocks. A–C: Limiting dilution assay with qPCR or QuantiGene 1.0 measurements. β-Actin measurements in purified RNA (pRNA) or TH from sample 23. Measurements of β-actin were conducted by qPCR (A) or bDNAFFPE assay QuantiGene 1.0 (B and C) in a serial dilution of the sample. The starting material is 100 ng of purified RNA or 1 μl of TH, which is the volume from which approximately 100 ng of RNA is extracted. Therefore sample inputs of pRNA and TH and subsequent linear dilution series are comparable. The concentrations of the serial dilution are indicated on the x axis in a log2 scale. The y axes in B and C are log2 relative luminescence units (log2RLU) of data obtained in the bDNA assay to facilitate comparison to qPCR. Thus the amounts of analyte indicated in the x axis are comparable between the three panels. In each panel, the LOQ is indicated by a vertical line. The LOQ in A is at 33 cycles, which is the conventional definition of the LOQ for this PCR machine. In panels 2 and 3, the LOQ is at the inflection point of the linear line, which indicates the point at which further dilution does not result in a proportional decrease in signal intensity. D: Limit of quantification comparing pRNA and TH in the QuantiGene 1.0 Reagent System. Two archival blocks, 23 and 58, were used to generate pRNA or TH. The limit of quantification for β-actin, RPL32, and SDHA was determined for each sample using the limiting dilution approach described above. Dilutions of sample input that provided quantifiable results are shaded in the graph. The end of the shaded bar represents the LOQ for each sample/assay/gene combination. The Journal of Molecular Diagnostics 2008 10, 169-176DOI: (10.2353/jmoldx.2008.070127) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Relative gene expression in cancerous versus normal prostate epithelium. A and B: Assessment of RNA quality and amount. A: Three sections from archival tissue blocks were solubilized in tissue homogenizing solution. Measurements of 28S rRNA (10 μl) were performed with the QuantiGene 1.0 Reagent System in a 105-fold dilution of the original sample. Measurements of 18S rDNA (10 μl, 102-fold dilution) are proportional to cell number in the sample and were conducted with the QuantiGene 1.0 Reagent System in the same TH. B: The amount of measurable 28S rRNA was normalized to 18S rDNA for comparison of RNA quality between samples. Samples that provided 28S rRNA measurements above the limit of detection in the bDNA assay, defined as 3 SD above background, were used for gene expression measurements. C: Measurement of a cancer gene panel with the QuantiGene 2.0 Reagent System. Cancer and normal tissues from the same blocks were macrodissected and dissolved in homogenizing solution. A panel of 14 prostate cancer genes were measured in cancer and normal tissues using the bDNA QuantiGene 2.0 amplification system. Values for each gene were normalized to RPL19 as a housekeeping gene. The ratio between cancerous and normal tissues is calculated and shown in a five-tiered categorical scale. The Journal of Molecular Diagnostics 2008 10, 169-176DOI: (10.2353/jmoldx.2008.070127) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions