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miRSIGN miR-21 Oncogene Assay (RUO)

Fast, easy and robust qPCR-based detection of hsa-miR-21-5p in colon carcinoma from formalin-fixed, paraffin embedded (FFPE) human colon tissue specimens. Contains all reagents needed for accurate detection of miR-21. Samples are analyzed for the expression level hsa-miR-21 relative to two stably expressed reference microRNAs.


NOTE: Although high levels of miR-21 are indicative of malignancy, this assay is designed for Research-Use-Only and is not to be used in a clinical setting.
  • Fast and easy PCR-based measurements of the miR-21 oncogene in FFPE samples
  • Based on Exiqon’s robust miRCURY LNA™ Universal PCR microRNA PCR System which offers excellent and reliable results from FFPE samples
  • Excellent sensitivity and specificity
  • Contains assays for reference genes stably expressed across a wide range samples and disease stages

Features

The miRSIGN miR 21 Oncogene Assay (RUO) detects the amplification level of hsa-miR-21-5p in colon carcinoma using the miRCURY LNA™ Universal RT microRNA PCR system on RNA extracted from FFPE human colon tissue specimens. Samples are analyzed for the expression levels of hsa-miR-21-5p relative to two reference microRNAs.

Content

The Kit contains all reagents required for performing the miRSIGN miR-21 Oncogene Assay (RUO) using total RNA extracted from FFPE tissue samples. The kit contains reagents sufficient for testing 120 samples (480 reactions):

Universal cDNA Synthesis Kit II, 8-64 rxns (2 kits)
ExiLENT SYBR® Green master mix, 2.5ml, 500 rxns (1 kit)
hsa-miR-21-5p LNA™ PCR primer set, UniRT
hsa-miR-16-5p LNA™ PCR primer set, UniRT
hsa-miR-103a-3p LNA™ PCR primer set, UniRT

The test is developed for RNA extracted from FFPE tissue blocks with a tissue area of > 4 mm2 or one unstained 10 µm slide.

Application

Recent research has shown that microRNAs may be responsible for the regulation of up to 40% of all oncogene expression. Because of their involvement in pathway regulation and other biological functions, microRNAs show great potential for use as diagnostic markers. Furthermore, clinical studies have found associations between microRNA expression patterns and treatment outcomes for several cancers, including colon cancer. The miRSIGN miR 21 Oncogene Assay (RUO) is highly reproducible (see experimental data) and based on the most sensitive and specific microRNA qPCR system available according to the miRQC study published in Nature Methods.

Read more about the miRCURY LNA™ Universal RT microRNA PCR system.

miRSIGN miR 21 Oncogene Assay (RUO)

In 2012, a study using Exiqon's proprietary LNA™-based miRCURY LNA™ microRNA Detection probes for in situ hybridization (ISH) to analyze the expression level of microRNA-21 (hsa-miR-21-5p) was published in British Journal of Cancer (ref 6). The study showed that high expression of hsa-miR-21-5p was associated with significantly poorer recurrence-free cancer-specific survival in a cohort of 554 patients with stage II colon cancer. The finding is supported by other studies (ref 2-6) and suggests that analyses of hsa-miR-21-5p should be considered as a potential adjunct in the selection of high risk stage II colon cancer patients.

The ISH assay has been migrated to the Exiqon's microRNA qPCR platform, miRCURY LNA™ Universal RT microRNA PCR, resulting in the miRSIGN miR-21 Oncogene Assay (RUO) kit. The prognostic value of the miRSIGN miR-21 assay (RUO) was evaluated in a study published in British Journal of Cancer 2014 (ref 1).

The miRCURY LNA™ Universal RT microRNA PCR system which combines a universal RT reaction with microRNA specific LNA™-enhanced primers enabling unmatched sensitivity and microRNA quantification (Figure 1a). By using the same RT reaction as template in all subsequent PCR reactions, the number of pipetting steps is reduced to a minimum thereby minimizing the technical variation achieving an extremely reproducible system even from site-to-site (Figure 1b). These features in combination provide the miRSIGN miR-21 Oncogene Assay (RUO) being very robust with a high level of accuracy and reproducibility.

The study applied the miRSIGN miR-21 Oncogene Assay (RUO) for investigation of the expression level of hsa-miR-21-5p on the same population-based cohort of 554 patients with stage II colon cancer, again aiming at generating a risk index for this group of patients. The study confirmed that high hsa-miR-21-5p expression was associated with an unfavorable recurrence-free cancer-specific survival with a hazard ratio 1.35 (95% CI, 1.03–1.76, P<0.028).

The study supports hsa-miR-21-5p as an independent prognostic biomarker in patients with stage II colon cancer. Further, inclusion of hsa-miR-21-5p in the risk index for stratifying patients with stage II colon cancer may guide the use of postoperative adjuvant treatment in a more appropriate way compared with current practice. In this study, inclusion of hsa-miR-21-5p in the risk index results in identification of 23% of the patients as being at high risk compared to 77% of the patients with the traditional risk parameters. The proposed risk index classifies patients correctly (high or low risk) with a probability of approximately 70%. The cross-validated AUC for the recurrence-free cancer-specific survival index at 1- and 5-year was 0.714 and 0.667, respectively.

The identification of high risk patients is a first step in a possible stratification of patients for adjuvant therapy. The current study does not address whether high risk patients with elevated microRNA-21 expression also will benefit from adjuvant therapy. The possible predictive value of hsa-miR-21-5p in identifying high-risk patients who will respond to adjuvant chemotherapy will require further clarification in a clinical context.

1. Hansen TF, Kjær-Frifeldt S, Christensen RD, Morgenthaler S, Blondal T, Lindebjerg J, Sørensen FB, Jakobsen A (2014). Redefining high-risk patients with stage II colon cancer by risk index and microRNA-21: results from a population-based cohort. Br J Cancer 111: 1285-1292
2. Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, Yanaihara N, Yuen ST, Chan TL, Kwong DL, Au GK, Liu CG, Calin GA, Croce CM, Harris CC (2008). MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA 299: 425–436
3. Kulda V, Pesta M, Topolcan O, Liska V, Treska V, Sutnar A, Rupert K, Ludvikova M Babuska V, Holubec Jr. L, Cerny R (2010). Relevance of miR-21 and miR-143 expression in tissue samples of colorectal carcinoma.
4. Shibuya H, Iinuma H, Shimada R, Horiuchi A, Watanabe T (2010). Clinicopathological and prognostic value of microRNA-21 andmicroRNA- 155 in colorectal cancer. Oncology 79: 313–320
5. Nielsen BS, Jorgensen S, Fog JU, Sokilde R, Christensen IJ, Hansen U, Brunner N, Baker A, Moller S, Nielsen HJ (2011). High levels of microRNA-21 in the stroma of colorectal cancers predict short diseasefree survival in stage II colon cancer patients. Clin Exp Metast 28: 7–38
6. Kjaer-Frifeldt S, Hansen TF, Nielsen BS, Joergensen S, Lindebjerg J, Soerensen FB, Depont CR, Jakobsen A (2012). The prognostic importance of miR-21 in stage II colon cancer: a population-based study. Br J Cancer 107: 1169–1174


Figure 1a Serial dilution of microRNAs constituting the miRSIGN miR-21 Oncogene Assay (RUO) proving high system sensitivity
Serial dilution of microRNAs constituting the miRSIGN miR-21 Oncogene Assay (RUO) proving high system sensitivity (Click to learn more)

Figure 1b High reproducibility of the microRNA signature assays in the miRSIGN miR-21 Oncogene Assay (RUO)
High reproducibility of the microRNA signature assays in the miRSIGN miR-21 Oncogene Assay (RUO) (Click to learn more)

A unique system developed specifically for microRNA profiling

The miRCURY LNA™ Universal RT microRNA PCR System offers the best available combination of performance and ease-of-use on the market because it unites two important features (Figure 2):
  • One cDNA reaction for all microRNAs – One single Universal first strand cDNA synthesis reaction is used as template, regardless of the number of microRNAs being profiled. This saves precious sample, reduces technical variation, means less pipetting and saves time in the laboratory.
  • Two LNA™-enhanced microRNA qPCR primers – Both qPCR primers are microRNA-specific and optimized with LNA™. LNA™ primers bind with high affinity, and are shorter than standard PCR primers, so that both primers can fit on the microRNA without overlapping. The result is unrivalled sensitivity and specificity, and extremely low background.

Unmatched sensitivity

Universal RT combined with LNA™-enhanced and Tm normalized primers enables accurate and reliable quantification of individual microRNAs from as little as 1 pg total RNA (Figure 3). In comparison to other microRNA real-time PCR systems using either stem-loop or standard DNA primers, the LNA™-enhanced primers offer significantly increased sensitivity, especially for AU-rich microRNAs.

Exceptional sensitivity as well as extremely low background enables accurate quantification of very low levels of microRNA without the need for pre-amplification. This makes the miRCURY LNA™ Universal RT microRNA PCR System suitable for all sample types, and especially samples with low RNA content e.g. biofluids such as serum/plasma.

Fully validated and optimized

All miRCURY LNA™ Universal RT microRNA PCR primer sets have been optimized for maximum sensitivity and thoroughly validated either by wet lab testing or in silico. In wet lab validation, over 95% of assays detect 10 microRNA copies or less in the PCR reaction (Figure 4). The primer sets have also been validated for specific amplification of the target and for minimal background signal.

Truly specific – no false positives

The incorporation of LNA™ in the qPCR primers facilitates the design of assays that can distinguish between microRNA sequences that differ by a single nucleotide. This makes it a truly specific microRNA qPCR platform – the only microRNA platform that gives no false positive signals (Figure 5). In addition, the assays can discriminate between mature microRNA sequences and precursor microRNA (Table 1).

Fast, easy and reproducible

Save time and effort in the laboratory with the 3 hour easy-to-follow protocol that minimizes pipetting. By using the same cDNA as template in all subsequent PCR reactions, the procedure is greatly simplified compared to systems that require microRNA-specific cDNA synthesis.

With fewer pipetting steps, technical variation is also reduced. As a result, it is possible to achieve extremely high reproducibility from day-to-day (Figure 6) and even site-to-site.

Flexible qPCR system

Tailor your experimental setup to your specific need using individual assays or fully flexible custom Pick-&-Mix panels . Using the same cDNA reaction, shift seamlessly between individual primers, pre-designed miRNome and Focus panels, or custom Pick-&-Mix panels.
Figure 1 Overview of the miRCURY LNA™ Universal RT PCR workflow
Overview of the miRCURY LNA™ Universal RT PCR workflow. (Click to learn more)

Figure 2 Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System
Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System. (Click to learn more)

Figure 3 Accurate quantitation from down to 1 pg total RNA starting material
Accurate quantitation from down to 1 pg total RNA starting material. (Click to learn more)

Figure 4 Serial dilution of hsa-miR-181a
Serial dilution of hsa-miR-181a. (Click to learn more)

Figure 5 Specificity results from the miRQC Study
Specificity results from the miRQC Study. (Click to learn more)

Table 1 Discrimination between mature and precursor microRNAs
Discrimination between mature and precursor microRNAs. (Click to learn more)

Figure 6 Excellent day-to-day reproducibility
Excellent day-to-day reproducibility. (Click to learn more)

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