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cDNA Synthesis and SYBR® Green Master Mix Kits

Optimized reagents for first strand cDNA synthesis and real-time PCR amplification using miRCURY LNA™ Universal RT microRNA PCR. ExiLENT SYBR® Green Master Mix offers improved sensitivity.

Nature Methods paper compares microRNA profiling platforms

In the largest peer-reviewed study of microRNA profiling platforms to date, Pieter Mestdagh and colleagues have compared the performance of commercially available systems in key areas.

Find out which system offers the best performance
  • Optimized reagents for use with the microRNA Ready-to-Use PCR panels and microRNA LNA™ PCR primer sets
  • NEW ExiLENT SYBR® Green PCR Master Mix offers improved sensitivity
  • Fast, easy and reliable first strand synthesis
  • Includes RNA spike-in template and Control Primer set for monitoring performance

Universal cDNA Synthesis kit II

All the reagents needed for fast and convenient microRNA polyadenylation and reverse transcription in a single reaction step. By using a single universal RT reaction as template in all subsequent PCR reactions, the procedure is greatly simplified compared to systems that require microRNA-specific first-strand synthesis (Figure 1).

When used in 20µL reactions, the kit contains reagents sufficient for 32 reverse transcription reactions. Reaction volumes can be varied between 10 and 80µL (Figure 2).

Contents of the kit:
  • 5x reaction buffer
  • 10x enzyme mix
  • nuclease-free water
  • RNA spike-in template (UniSp6)
The UniSp6 RNA spike-in can be used with the Control primer set provided with the ExiLENT SYBR® Green master mix or as a positive control and/or inter-plate calibrator in Exiqon’s microRNA PCR panels.

Please note, UniSp6 is supplied in double the amount of the old Universal cDNA Synthesis kit and should be dissolved in twice the amount of water to have the same working concentration. In all other regards the two kits are identical.

ExiLENT SYBR® Green master mix

ExiLENT SYBR® Green master mix is a high-performance PCR master mix kit specifically designed for Exiqon’s miRCURY LNA™ Universal RT microRNA PCR system.

The ExiLENT SYBR® Green master mix kit is required for use with Exiqon’s new v.4 miRCURY LNA™ Universal RT microRNA PCR assays and panels.

The kit is available in two sizes: a 2.5 mL kit containing reagents for 500 PCR reactions (10µL reactions) and a 20mL kit sufficient for 4,000 reactions.

Contents of the kit:
  • 2x ExiLENT SYBR® Green Master Mix
  • nuclease-free water
  • Control primer set (UniSp6 v2)
The Control primer set is designed for amplification of the synthetic RNA spike-in provided in the Universal cDNA synthesis kit. Please refer to the miRCURY LNA™ Universal RT microRNA PCR Instruction manual for details.


Figure 1 The benefit of a universal RT step
The benefit of a universal RT step. (Click to learn more)

Figure 2 Reagents needed for use with miRCURY LNA™ Universal RT microRNA PCR Panels and assays
Reagents needed per PCR panel. (Click to learn more)

Universal cDNA Synthesis kit II

The kit contains reagents for fast and reliable reverse transcription of all microRNAs into cDNA in a single reaction step. The number of pipetting steps are kept to a minimum in order to save time and minimize technical variation.

ExiLENT SYBR® Green Master Mix

This kit contains high performance reagents for quantitative real-time PCR amplification. The detection is based on SYBR® Green which allows quality control of the resulting PCR amplicon by melt curve analysis. LNA™-enhanced primer sets require specific PCR reaction conditions for optimal performance.
Exiqon's v.4 primer sets and PCR Panels have been validated using the ExiLENT SYBR® Green master mix. The use of other reagents for qPCR will affect the results.

A key feature with the ExiLENT SYBR® Green master mix is the remarkably low residual activity. This means it is possible to work reliably with Cq values up to 37 cycles, giving the platform exceptional sensitivity without the use of time consuming preamplification. Reliable profiling in the high Cq range is a key feature required for working with samples low abundant in microRNAs like biofluids (Figure 1).



Figure 1 Exiqon's qPCR system is ideal for challenging samples
Exiqon's qPCR system is ideal for challenging samples. (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)

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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