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miRSIGN Colon Cancer Test (RUO)

The miRSIGN Colon Cancer Test (RUO) provides a fast and easy method of determining whether or not colon cancer cells are present in formalin fixed paraffin (FFPE) embedded colon tissue specimens. Four microRNAs linked to colon cancer are profiled using Exiqon’s robust microRNA qPCR system and the resulting signature can be used to categorize the sample as tumor or normal.


NOTE: Although the signature is indicative of malignancy this test is designed for Research-Use-Only and is not to be used in a clinical setting.
  • Fast and easy PCR-based detection of colon cancer cells in FFPE samples
  • Ideal for tumor assessment where histological analyses provide unclear results
  • Based on Exiqon’s robust miRCURY LNA™ Universal PCR microRNA PCR System which offers excellent and reliable results from FFPE samples
  • Cancer-related microRNAs detected with unmatched sensitivity and specificity
  • 98% specificity in distinguishing colon cancer cells from normal cells

Features

The miRSIGN Colon Cancer Test (RUO) is based on the miRCURY LNA™ Universal RT microRNA PCR system and consists of LNA™ primer sets and accessory reagents required to analyze the expression levels of four key microRNAs known to be linked to colon cancer, in formalin-fixed, paraffin-embedded (FFPE) colon carcinoma tissue. This 4-miR signature provides a combined numerical expression level that is compared to a normal reference range and can be used to categorize the tissue sample as tumor or normal.

Content

The Kit contains all reagents required for performing the miRSIGN Colon Cancer Test (RUO) using total RNA extracted from FFPE tissue samples. The kit contains reagents sufficient for testing 100 samples (500 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-34a-5p LNA™ PCR primer set, UniRT
hsa-miR-126-3p LNA™ PCR primer set, UniRT
hsa-miR-143-3p LNA™ PCR primer set, UniRT

The test is developed for RNA extracted from FFPE tissue blocks with a tissue area of > 4 mm 2 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 Colon Cancer Test (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.


About the miRSIGN Colon Cancer Test (RUO)

The test detects the expression level of four microRNAs (hsa-miR-21-5p in combination with three other human microRNAs) which provides a numerical combined expression level that can be compared to a normal reference range and used to categorize the tissue specimens.

Internal validation studies have shown very high specificity of the test in distinguishing colon cancer specimens versus normal cancer specimens (98% specific in distinguishing colon cancer cells versus normal cells) (Figure 1).

The miRSIGN Colon Cancer Test (RUO) is based on Exiqon’s 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 2a). 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 2b). These features in combination have resulted in the miRSIGN Colon Cancer Test (RUO) – a very robust test with a high level of accuracy and reproducibility (Figure 3).
Figure 1 miRSIGN Colon Cancer Assay (RUO)
miRSIGN Colon Cancer Assay (RUO) Application (Click to learn more)

Figure 2a microRNA copies in PCR
microRNA copies in PCR (Click to learn more)

Figure 2b microRNA copies in PCR
High reproducibility of the four microRNA signature assays in the miRSIGN Colon Cancer Test (RUO) (Click to learn more)

Figure 3 Excellent run-to-run reproducibility
Excellent run-to-run reproducibility (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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