MELT™ Total Nucleic Acid Isolation System
As with many cancers, the molecular underpinnings of prostate cancer (PrCa) are poorly defined. Animal models, such as xenograft mice, offer an invaluable, in vivo context for studying both the etiology and progression of disease. Such models provide a path to cancer experimentation that can lend insights into the underlying biology and test the efficacy of new treatments. Recently, Ambion scientists began a collaboration with Drs. Jin-Rong Zhou and Sandra Gaston (Beth Israel Deaconess Medical Center, Harvard Medical School) and scientists at Illumina, Inc. to assess the global gene expression profiles in a mouse xenograft model for PrCa. An overview of their results is presented here.
Xenograft Mouse Model
Preparing RNA from Tumors
Figure 1. Integrity of RNA Isolated by MELT™ Total Nucleic Acid Isolation System. Total RNA (80–220 ng), isolated using the MELT Total Nucleic Acid Isolation System was run on an Agilent® 2100 bioanalyzer and is displayed here as a gel electropherogram. Samples 1 through 12 are the 12 biological replicates used in this research. RIN=RNA Integrity Number (10=high integrity, 1=low integrity)
Figure 2. Sample Signal Correlation by Illumina® Sentrix® Gene Expression Microarray. (A) Excellent signal correlation was observed between technical replicates. (B) Excellent signal correlation was generally observed between biological replicates, but decreased with advanced cancer progression. (C) This study contained 12 biological replicates, with technical replicates designated by an “a” or “b.” Green shading indicates inter-sample signal correlations above 0.990, orange shading indicates inter-sample signal correlations that are >0.980 and <0.990, and gray shading indicates inter-sample signal correlations that are <0.980.M
The array data were evaluated to identify genes with expression levels that varied at least 2-fold in androgen-independent vs. androgen-sensitive tumor tissue; nearly 50 genes met this criteria. The combination of the MELT Total Nucleic Acid Isolation System, Illumina TotalPrep RNA Amplification Kit , and Illumina Sentrix Array provided a powerful solution from sample-to-data to identify candidate RNA biomarkers of PrCa. A manuscript describing these results in full detail is in preparation.
Because of the relatively high RNase activity in the tumor implant samples in this study, samples were processed in small batches (six at a time), and the MELT digestion time was rigorously controlled (10 min) to minimize variability and maximize RNA yield. All lysates were stored overnight at –20°C, and RNA was recovered the following day in batches of 12–16 samples.