2014

Specific Detection of RNA Expression and Distribution in Brain Using RNAscope® Technology

Detect RNA with ease

• Single-molecule detection with single-target specificity by proprietary probe design and signal amplification technology
• Visualize RNA expression and distribution with morphological content
• Easy and robust Assay

Detection of Neurological Biomarkers

Understanding the molecular mechanisms of neurological disease development and progression is necessary to identify key pathogenic steps, and thereby identify new potential therapeutic targets through a combination of epidemiology-related neuropathology, biomarkers for clinical trials, and experimental studies. RNAscope^® in situ hybridization (ISH) technology enables cell- and tissue-specific localization of RNA transcripts quickly and precisely for functionally important targets in the nervous system. With RNAscope ISH assays, you can turn any gene specifically expressed by a particular cell lineage into an endogenous marker for neuronal cell types and tissues.

Visualize any RNA

RNAscope ISH technology achieves high sensitivity and specificity for detection of gene expression in the nerves system. For example, RNAscope-based CISH assay confirms that Apoe4, one of the transcript isoforms of ApoE, is locally expressed in the astrocytes of mouse brain (Figure 1); and Bmp1 expression is restricted to the grey zone of cerebella. RNAscope ISH is ideal for staining any RNA transcript in FFPE (Figure 1) or fresh frozen (Figure 2) neurological tissues, and can provide in situ quantitative analysis for any gene in any species. RNAscope ISH is especially powerful in detection of low-abundance RNA, as well as targets to which there are no good antibodies available.

FIGURE 1. Apoe4 mRNA expression in mouse brain FFPE tissue using the RNAscope® single-plex Chromogenic Reagent Kit - BROWN

FIGURE 2. Polr2a (green), Ppib (white), Ubc (red) in mouse brain fresh-frozen tissue using the RNAscope® Multiplex Fluorescent Assay

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Assess Transcriptionally Active HPV Biomarkers in Head and Neck Cancer Biopsies

Detect HPV E6/E7 mRNA expression with tissue morphological context

• High Sensitivity and Specificity—single molecule detection with single-target specificity by proprietary probe design and detection technology
• Broadest Spectrum—with flexibility of individual genotyping, pooled high-risk and low-risk panels (HPV-HR7, HPV-HR18, HPV-LR6) and custom pooled panels
• Robust and Easy Methodology—scalable with automation for routine analysis

Detection of Gold-standard E6/E7 Oncogene Transcripts Using RNAscope® ISH

Evidence for transcriptional active of the viral oncogenes E6/E7 is regarded as the gold standard for presence of clinically relevant high-risk human papillomavirus (HPV), but detection of E6/E7 mRNA can be challenging using conventional techniques (Bishop et al., 2013). As a causal agent in head and neck squamous cell carcinoma (HNSCC), it is critical that the detection method enable pathologist review of tissue morphology and be of the highest specificity and sensitivity for accurate assessment of within the tissue microenvironment of FFPE specimens (Figure 1, 2). RNAscope HPV Biomarker Detection Reagents and its proprietary “double Z” oligonucleotide probes specific for each subtype E6/E7 mRNA enable high specificity detection of viral transcripts in routine FFPE tumor biopsies.

FIGURE 1. Schematic illustration of the Biology of HPV infection provides points of HPV Detection (Bishop et al). Testing E6/E7 transcripts by RNA ISH is desirable because it indicates the presence of transcriptionally active virus and enables visualization of viral transcripts directly in tumor cells in tissue sections, unlike RT-PCR (Upko et al, 2011).

FIGURE 2. High risk HPV E6/E7 mRNA expression in Head and Neck Squamous Cell Carcinoma (HNSCC). FFPE section of HNSCC hybridized with a pool of HPV 16, 18, 31, 33, 35, 52 and 58 genotype probes, showing cytoplasmic punctate dots only in the tumor cells (40X magnification).

Highest Sensitivity and Specificity

Current methodologies for HPV testing include PCR-based amplification and DNA in situ Hybridization (ISH). PCR amplification of HPV DNA is more sensitive, but it is less specific than DNA ISH. Published studies (Bishop et al., 2013, Upko et al., 2011, and Schache et al., 2013) indicate that RNAscope-based ISH assay is more sensitive than DNA ISH in detecting HPV in Oropharyngeal Squamous Cell Carcinoma (OSCC) and results correlate well with p16 immunohistochemistry (IHC) staining (Table 1). RNAscope ISH technology is an ideal platform giving the high sensitivity and specificity needed for detection of HPV biomarkers in Head and Neck Squamous Cell Carcinoma (HNSCC) samples.

TABLE 1. Using the current “gold-standard” qPCR as the reference method, RNAscope® Technology demonstrated the best sensitivity and specificity for HPV status determination than existing methods (Schache et al., 2013). Abbreviations: IHC=immunohistochemistry; ISH=in situ hybridization; NPV=negative predictive value; PPV=positive predictive value; qPCR=quantitative PCR

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Pure Water for the Modern Clinical Laboratory

Summary

Clinical grade water is clearly essential in a clinical laboratory and the trend is certainly towards higher purity requirements to meet the range of analyses carried out. Poor water quality not only affects the tests themselves directly but also impacts on all aspects of analyzer operation. Good water purification design, especially providing recirculation through the key purification technologies, is the key to effective and long-term bacterial control, which is the most challenging aspect of water purification. This approach, combined with duplex operation and good support will provide the water needed.

Introduction

Water purity has always been important in clinical diagnostics but on-going developments in the approach to clinical testing and in the sophistication and range of tests available have made water purity even more critical. The key requirements for water for clinical analyzers are:

• Highly reliable, uninterrupted supply
• Consistent high purity
• Low running costs
• Easy operation and minimal user intervention

Traditionally, analyzer feed water systems were mainly for chemistry-based analyzers that used principally colorimetric and ion selective electrode technologies. The overall trend towards less invasive technologies has led to smaller blood and other samples and their use for a wider range of tests. 500μl samples have to provide material for the initial tests and with some in reserve for retests or further studies. This has become feasible with improvements in pipetting techniques which enable high accuracy at volumes of only a few micro-liters. Sample dilution with clinical grade water prior to assay puts extremely high demands on the water purity as the effects of any contaminants are immediately multiplied by the dilution factor. Limited samples may also restrict scope for retesting, adding extra clinical pressure to the financial pressures of ensuring that reliable results are obtained first time.

Uses of Clinical Grade Water

Figure 1: Uses of Pure Water

Increasingly, automated immunoassay systems have been introduced into routine clinical chemistry labs. These tend to use disposable cuvettes and sample pipette tips but still need pure water for reagent probe tip washing, pipetting and dilution of bulk reagents. Generally a higher quality of water is required for immunoassay because of the highly sensitive technologies used, such as chemiluminescence, either direct or indirect, or time-resolved enzymatic fluorescence. Many tests rely on multi-stage processes using enzymes such as alkaline phosphatase. Any drop in water purity can seriously impact on the quality and reliability of these assays. Currently automation within clinical chemistry labs has progressed to group all the high volume tests together onto common platforms irrespective of the measuring technology. It is fairly commonplace now to use systems like the Abbott Diagnostics ARCHITECT^® ci8200 and the Roche cobas^® 6000 platform, both of which combine immunoassay and chemistrybased tests. The water feeding these systems tends to be used by both types of technology, and so all the water has to be pure enough for the more sensitive immunoassay technologies.

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