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RNA Spike-In Kit

RNA spike-in kit for quality control of the RNA isolation and cDNA synthesis steps of a miRCURY LNA™ Universal RT microRNA PCR experiment.

Biofluid sample QC and identification of hemolyzed samples

Read more about qPCR-based sample QC and Exiqon’s unique hemolysis indicator.

Download Biofluids Guidelines
  • An easy and convenient control of your RNA samples and RT reactions
  • Saves time and reagents by allowing you to identify poor samples early
  • Available in an easy-to-use pre-mixed format
  • Highly flexible – Use only the spike-ins you need for your study

Features

Use the RNA spike-in kit for quality control of the RNA isolation and cDNA synthesis steps of a miRCURY LNA™ Universal RT microRNA PCR experiment. It is important for any qPCR experiment to ensure that the quality of the input RNA is sufficiently high for effective amplification. By introducing spike-in RNAs during RNA isolation and cDNA synthesis and comparing the qPCR results to those from endogenous reference genes, important information regarding the quality of the samples can be obtained (Table 1).

Table 1. Overview of issues and conclusions


Product coverage

Exiqon’s RNA spike-ins (UniSp2, UniSp4, UniSp5, UniSp6) resemble microRNAs in structure but lack close sequence similarities to known microRNAs. In addition, the kit includes a synthetic version of the C. elegans microRNA, cel-miR-39-3p.
The RNA Spike-in kit is distributed in two vials:
  • Vial 1: Contains 3 RNA spike-ins (UniSp2, UniSp4 and UniSp5) pre-mixed, each at different concentration in 100 fold increments (Table 2). This set of spike-ins is intended as an RNA isolation control.
  • Vial 2: Contains a synthetic version of cel-miR-39-3p. This microRNA is meant to be used in conjunction with the UniSp6 spike-in provided with the Universal cDNA Synthesis Kit (denoted “RNA spike-in”). Mixing of these RNA templates as described in the manual will result in a 100 fold concentration difference between the RNAs (Table 2). This set of spike-ins is intended as a cDNA synthesis control.
Each vial contains RNA sufficient for 50 reactions. In addition, they contain MS2 carrier RNA intended to improve the stability of the RNA spike-ins without interfering with down-stream reactions.

Table 2. Contents of the RNA Spike-In Kit


Choose the spike-in RNAs you prefer

In order to take advantage of the RNA Spike-in kit, you will need to purchase the corresponding LNA™ primer sets separately (with the exception of UniSp6 which is included in Exiqon’s SYBR® Green Master Mix). This means that you have the flexibility to use only the spike-ins you need and don’t have to pay extra for primer sets you are not going to use.

Compatibility

The RNA Spike-in Kit is compatible with Exiqon Pick-&-Mix microRNA PCR Panels and individual miRCURY LNA™ Universal RT microRNA PCR assays.

Analysis and interpretation of data

An important factor when using the RNA Spike-In Kit is knowing how to interpret the experimental data. Exiqon has made this easy by devoting an entire section in the manual to explaining how to analyze and interpret the data. Learn how to analyze your data in an easy-to-understand clear-cut way: Download the RNA Spike-In manual

Figure 1 Exiqon’s RNA Spike-In Kit covers the full range of microRNA expression levels
Exiqon’s RNA Spike-In Kit covers the full range of microRNA expression levels. (Click to learn more)

View experimental data on miRCURY™ microRNA QC PCR Panel.

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)

NOTICE TO PURCHASER: LIMITED LICENSE Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. SYBR® Green is a trademark of Invitrogen.
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