MELT™ Total Nucleic Acid Isolation System
Cancer Biomarker Quantification Using RNA Extracted from Tumor Tissue Print Micropeels

Dr. Sandra Gaston and colleagues (Beth Israel Deaconess Medical Center) described the application of tissue print “micropeels” to clinical specimens as a way to quickly and easily capture molecular information from human tissue samples prior to fixation and subsequent histopathological analysis [1]. In clinical practice, tissue specimens are usually fixed and embedded as standard procedure for the preparation of pathology slides; however, this procedure can severely damage cellular RNA and compromise RNA expression patterns. In contrast, tissue print micropeels can preserve biological information from the sample with minimal intrusion to the pathologist’s workflow and without impacting the downstream histopathology of the sample. Moreover, sufficient RNA and protein biomarkers can be quantitated within tissue prints to detect expression signatures consistent with the presence of disease in the underlying tissue.

Isolating RNA from Tissue Prints Captured on Nitrocellulose
One problem with conventional RNA isolation from tissue prints (see sidebar, What is a Tissue Print Micropeel?), is that the RNA can be difficult to release into a standard GITC extraction buffer unless the tissue print is vigorously scraped and agitated for at least 10 min to get all of the bound cells into suspension. To address this problem, Dr. Gaston and Ambion began a collaboration to determine if the enzymatic strategy offered by the MELT™ Total Nucleic Acid Isolation System might expedite the recovery of RNA. It was found that MELT offers a much simpler procedure (e.g., less hands-on time, 1 min MELT incubation) for extracting RNA from tissue prints, without any loss in yield (data not shown). Subsequently, a tissue print-MELT protocol was developed (Figure 1), and this procedure was used to evaluate the presence of cancer biomarkers from tissue prints taken from a radical prostatectomy clinical sample containing a localized tumor.

Figure 1. RNA Isolation From Tissue Print Tiles Using MELT™ Total Nucleic Acid Isolation System.

Case Study: Identifying Prostate Cancer Biomarkers
Slices from a surgically resected prostate specimen were printed using a sheet of nitrocellulose that covered both the tumor and benign adjacent tissue regions. The print was then carefully sliced into tiles to create a spatial map that represented both normal and cancerous tissue areas [1]. A small (4 x 4 mm) tile from either the normal or tumor region was added to the MELT™ reagent, and total RNA isolated. The purified RNA was then analyzed by quantitative RT-PCR using Ambion’s MessageSensor™ RT Kit. Five transcripts were assayed: 18S rRNA, a normalization control; B2M (b-2-microglobulin), a representative housekeeping gene; and three well-documented RNA biomarkers that are known to be upregulated in prostate cancer compared to normal tissue (AMACR, Hepsin, and PCA3) [2, 3]. As shown in Figure 2, all three biomarkers were significantly more highly expressed in the tissue print tile corresponding to the location of the underlying tumor (confirmed by H&E staining) compared to tiles from benign prostate regions from the same patient. In contrast, the housekeeping genes 18S and B2M, were expressed equally, suggesting that approximately the same number of cells were extracted from each tile. Thus, MELT sample preparation of tissue prints from human tissue specimens can recover disease-relevant biomarkers that can “map” the location of cancerous cells.

Figure 2. Identification of Biomarkers from a Prostate Tissue Print. Quantitative RT-PCR was used to analyze total RNA (1 ng) isolated from two prostate tissue print tiles using the MELT™ Total Nucleic Acid Isolation System. PCR targets included 18S rRNA, b-2-microglobulin (B2M), and prostate cancer biomarkers (AMACR, Hepsin, and PCA3). Error bars indicate the difference between triplicate reactions. DDCt = [Ct-gene–Ct-18S rRNA]Normal–[Ct-gene–Ct-18S rRNA ]Tumor

Scientific Contributors
Gary Latham and Amber Magotra • Ambion, Inc.
Sandra M. Gaston • Department of Surgery, Division of Urology, Beth Israel Deaconess Medical Center

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