Ligo-miR EZ Assay Kit

Future Ligo-miR Tools

Ligo-miR EZ 19+5 Assay Kit*

The Ligo-miR EZ assay kit contains of all probes, enzymes, and buffers necessary to perform 24-plex microRNA profiling on 50 samples. Multiplex analysis of 24 microRNA is accurately performed in each reaction using our innovative PCR-free, ligation assay.

Ligo-miR EZ Assay Kit

The assay is performed in 3 simple steps. First, the capture ligation reagents are added to an RNA sample and incubated for 1 hour. Second, the coding ligation reagents are further added to the mixture and thermal cycled  for 50 cycles. Finally, the reaction mixture is analyzed by PAGE. Each lane represents a single sample where each microRNA is accurately identified by band position and expression level is precisely determined by band intensity. With this simple method Ligo-miR EZ achieves sensitivity 100-1000x higher than microarray!

Ligo-miR EZ Process

Ligo-miR EZ 19+5 uses a semi-custom panel consisting of 19 fixed microRNA and 5 user selectable microRNA. The 19 fixed microRNA comprise 5 control microRNA (3 spike-in + 2 snoRNA) and 12 application specific microRNA (e.g. general cancer, breast cancer, cardiovascular, etc). The 5 variable microRNA are selected at the user's discretion to enable application specific tailoring. Users will then be sent a kit containing their semi-custom 19+5 panel. Fully custom panels can also be created.

High Sensitivity1

  • 2.5e-18 moles sensitivity using fluorescent PAGE detection (no PCR)
  • 50X linear amplification
  • Linear quantification across 3-4 orders of magnitude dynamic range
  • 1,000X higher sensitivity than microarray
  • Sensitivity rivals Nanostring and Illumina
 

Ligo-miR mir-21 Titration

Fig 1. Linear quantification down to 2.5e-18 moles ± 20% exp-exp CV.

Low Bias

  • High efficiency and low bias microRNA capture
    • 86% (AVG) ± 10% (SD) efficiency across across a representative 20 microRNA panel
    • No adapter pools necessary
    • Flex couple into other microRNA methods
  • Linear amplifcation = low amplification bias
  • Highly reproducible
    • Exp-Exp detection efficiency CV ~10-15%
  • High correlation to qPCR and microarray profiles
 

Ligo-miR EZ miRNA Capture

Fig 2. 20 microRNA panel captured at 86% (AVG)±10% (SD) efficiency.

High Specificity

  • No cross-talk across unrelated microRNA
  • Absolute discrimination of mature vs. pre-cursor microRNA
  • High specificity discrimination of closely-related microRNA families
    • Discriminates single nucleotide differences
 

Lig-miR EZ Let-7 family

Fig 3. High specificity discrimination of let-7 family.

High Sample Throughput

  • To screen 96 samples with 24 microRNA read depth:
    • Ligo-miR EZ - 1 well plate / 4 hours total time
    • PCR - 24 well plates / 8 days total time2
    • Microarray - 96 microarrays / 8 days total time3
    • Nanostring - 8 cartridges / 3 days total time4
  • 20X Reduction in Time Compared to Next Fastest Product
 

ligomir 6-plex

Fig 4. No cross-talk across unrelated microRNA. Absolute discrimination of mature vs. precursor

Lower Assay Costs and Labor1

  • For each 24 microRNA test:
    • Ligo-miR EZ - Only $20/sample
    • PCR - 24 individual reactions costs $104/sample
    • Microarray and Nanostring - >$220/sample
    • 24X less pipetting than qPCR
  • ~10-20X Cost Savings

Reduced Capital Investments

  • Ligo-miR EZ can be performed using only a thermal cycler and gel apparatus
  • Nanostring and Illumina platforms require exotic instrumentation (>$200k).
 

ligomir panel

Fig 5. Raw PAGE image of 6-plex microRNA expression data. Each of the 4 lanes contains expression data for a single sample. Each band position represents a single microRNA with band intensity representing expression level.

 

*Patent Pending
1Specifications and prices from http://www.lifetechnologies.com/, http://www.nanostring.com/, http://www.nature.com/app_notes/nmeth/2007/071601/full/an2256.html, Digital Multiplexed Gene Expression Analysis Using the Nanostring nCounter System in Current Protocols in Molecular Biology: John Wiley & Sons, Inc, and Direct Multiplexed Measurement of Gene Expression with Color-Coded Probe Pairs. Nature Biotechnology v26, p317-25.
2Assumes a single PCR machine, 4 hours per reaction, 3 reactions per day
3Assumes 12 hybridization chambers, overnight hybridization,
4Assumes cartridges run 24h a day