Quantitative RT-PCR (qRT-PCR) is the standard for validating microarray data and is an invaluable tool for highly sensitive and accurate profiling of miRNA population subsets. Most commercially available miRNA qRT-PCR systems use proprietary, predesigned, miRNA-specific primers for real-time PCR and/or reverse transcription. Unfortunately, such primers require that the miRNA sequence is publicly available, and that a commercial qRT-PCR assay has been developed for that specific sequence. This limits the availability of qRT-PCR assays for many model organisms, recently discovered miRNAs or noncoding RNAs, or proprietary miRNA sequences.

The NCode™ VILO™ miRNA cDNA Synthesis and NCode™ EXPRESS SYBR® GreenER™ miRNA qRT-PCR Kits overcome these limitations by combining a carefully optimized combined polyadenylation and  reverse transcription step using SuperScript® VILO™ and a universal primer. The miRNA-specific amplification occurs during the PCR reaction, in which the sequence of the miRNA of interest is used as the target-specific PCR primer.

Use the NCode™ EXPRESS SYBR® GreenER™ miRNA qRT-PCR Kits to:

  • Use minimal total RNA input (no additional enrichment needed, conserve precious samples) (Figure Y)
  • Detect miRNA, ncRNA, or mRNA sequences from the same universal cDNA
  • Archive cDNA for later validation—the use of the same total RNA sample for experiments over time will ensure more reliable comparison and allow future interrogation of sequences not yet discovered
  • Obtain excellent sensitivity over a broad dynamic range of miRNA abundance
  • Profile closely related miRNAs with single-nucleotide discrimination
  • Use multiple real-time PCR instrument platforms, including standard and fast-cycling modes

Figure Y— miRNA detection from total RNA or enriched miRNA starting materials.

cDNA was generated from brain reference RNA or an equivalent volume of enriched miRNA. Average Ct values for 10 ng/ul total RNA or equivalent per qPCR reaction are shown.

Figure Y Table

microRNA Total RNA Enriched RNA
26a16.5316.53
19517.1917.28
124.9725.36

Match the NCode™ qPCR tool to your research needs:

NCode™ EXPRESS SYBR® GreenER™ miRNA qRT-PCR Kits–incorporate proven Invitrogen™ enzymes and the newest SYBR® GreenER™ dye now specifically tailored to deliver superior results on high-throughput, fast-cycling instruments. These kits provide:

  • Fast-activating, antibody-mediated Platinum® Taq DNA Polymerase in association with the brighter and less PCR-inhibitory SYBR® GreenER™ dye—complete, rapid activation provides the best sensitivity and reproducibility
  • Flexible format with ROX premixed or separate—designed to work with default instrument protocols on a wide range of high-throughput fast instruments ( e.g., AB 7500 FAST, AB 7900 HT Fast, AB StepOne, Roche LightCyler® 480, Corbett Rotor-Gene™
  • Significantly reduced carryover contamination with UDG carryover protection—uracil-DNA-glycosylase (UDB)/dUTP incorporated in all kits

NCode™ VILO™ miRNA cDNA synthesis Kit—provides reagents only for the first-strand cDNA reaction allowing you to use your own optimized qPCR supermix. Tried and tested SuperScript® III Reverse Transcriptase is formulated in an enhanced buffer system to provide you with the most reliable first-strand synthesis and higher cDNA yields.

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