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ExiSEQ LNA™ Quant kit

A unique probe-based system for the most accurate quantification of libraries prior to RNA or DNA Next Generation Sequencing on Illumina® instruments. Ensure equal loading and optimal sequencing results.

  • Quantifies mRNA, whole transcriptome, microRNA or DNA libraries for the Illumina® platform
  • The most accurate quantification – the only probe-based system that counts molecules
  • No need to correct for library fragment size
  • Ensures equal loading of samples and optimal sequencing results
  • High performance LNA™ probe and LNA™ primers designed by Exiqon's LNA™ experts
  • Provided in a tube or in ready-to-use PCR panels for easy set up and high reproducibility

The only probe-based system for quantification of Illumina® libraries

The ExiSEQ LNA™ Quant kit is the most accurate way to quantify RNA or DNA libraries prior to Next Generation Sequencing on the Illumina® platform. The ExiSEQ LNA™ Quant kit is the only system for quantification of libraries for Illumina® sequencing that is based on absolute quantification using a dual-labeled hydrolysis probe (Figure 1). Library quantification is accomplished following an easy and rapid workflow (Figure 2).

Accurate library quantification is crucial for optimal sequencing results

Next Generation Sequencing typically involves pooling several different barcoded libraries together. Before pooling, the libraries are quantified and this information is used to ensure that the libraries are pooled together in equal amounts and the optimal amount of library is loaded onto the flow cell for cluster generation.

Maximize the number of reads and analysis power

If the libraries are not pooled in equal amounts, more reads will be obtained for some samples than for others. The power of the data analysis is limited by the sample with the fewest reads. Therefore, accurate library quantification is essential to ensure equal sample loading, thus maximizing the number of reads obtained and the power of the data analysis.

Ensure optimal data quality

Accurate library quantification is critical to obtain high quality sequencing data. Loading too little of the pooled library onto the flowcell can lead to low cluster density and a reduced sequencing yield. On the other hand, loading too much can increase the cluster density. When too many clusters are formed, they start to blend together at the borders, which decreases data quality.

More accurate library quantification than SYBR® Green

The challenge with library quantification is that fragment length can vary, depending on sample quality for example. All other methods for quantification of libraries for Illumina® sequencing use SYBR® Green which quantifies the template mass, so signal will vary depending on the length of the fragments in the library. Therefore, when using SYBR® Green, it is necessary to estimate the size distribution of the library and adjust the abundance estimates accordingly, which results in less accurate library quantification and often unequal pooling of samples.

Count molecules – no need to estimate library size distribution

The ExiSEQ LNA™ Quant kit is probe-based, so the signal from one probe equates to one molecule. Highly accurate absolute quantification is obtained using a standard curve generated from included DNA standards (Figure 3). So it is not necessary to correct for any variation in the size distribution of the fragments in the library. A library concentration calculator is provided here.

High performance LNA™ probe and LNA™ primers

The LNA™ probe and LNA™ primers are optimized by Exiqon's LNA™ experts for highly accurate quantification of libraries for the Illumina® platform. LNA™ primers target the P5 and P7 Illumina® adaptor sequences and the dual-labeled LNA™ probe targets the Index I (i7) sequencing primer binding site (Figure 1).

Compatible with all libraries for Illumina® platform with i7 index

The ExiSeq LNA™ Quant kit can be used for quantifying mRNA, microRNA and DNA libraries prepared for sequencing on Illumina® instruments. The ExiSEQ LNA™ Quant Kit is compatible with library preparation kits containing the primer for the Index I primer binding site (i7) including:
  • QIAGEN® NGS kits for Illumina® platform (not smallRNA kit)
  • Illumina® TruSeq based libraries (not Nextera™ transposase-based kits or smallRNA kits)
  • Agilent® NGS kits for Illumina® platform (not SureSelectQXT transposase-based kits)
  • Bioo Scientific NGS kits for Illumina® platform
  • Nugen NGS kits for Illumina® platform

Contents of the kits

The LNA™ primers and probe and are provided in a tube or in ready-to-use PCR panels for maximum ease of use and reproducibility (Figures 4 and 5). The PCR panels are compatible with all major PCR instruments. Plate layout files for the 96-well ready-to-use PCR panels are available to download.

ExiSEQ LNA™ Quant Kit, 200rxn:
  • LNA™ Quant primers and probe mixture, 200 rxns
  • Four LNA™ Quant Standard Templates (20 pM, 2 pM, 200 fM, 20 fM), 60 rxns each
  • Dilution buffer
This kit is sufficient for 200 reactions or 61 samples, 2 dilution curves and two No Template Controls, all performed in triplicate.

ExiSEQ LNA™ Quant Kit, panel:
  • Two LNA™ Quant 96 well plates with dried down primers and probe
  • Four LNA™ Quant Standard Templates (20 pM, 2 pM, 200 fM, 20 fM), 60 rxns each
  • Dilution buffer
This kit is sufficient for 192 reactions or 2x 27 samples, 2x dilution curves and two No Template Controls, all performed in triplicate.

Note: We recommend the use of QuantiTect Probe mastermix PCR kit (Cat. No./ID 204343, QIAGEN® Inc.) together with the ExiSEQ LNA™ Quant Kit.

Figure 1 A unique probe-based system for library quantification.
A unique probe-based system for library quantification. (Click to learn more)

Figure 2 A rapid, easy workflow for library quantification.
A rapid, easy workflow for library quantification. (Click to learn more)

Figure 3 Accurate absolute quantification of libraries by molecule counting.
Accurate absolute quantification of libraries by molecule counting. (Click to learn more)

Figure 4 Overview of the ExiSEQ LNA™ Quant Kit.
Overview of the ExiSEQ LNA™ Quant Kit. (Click to learn more)

Figure 5 Ready-to-use PCR panels for maximum ease of use and reproducibility.
Ready-to-use PCR panels for maximum ease of use and reproducibility. (Click to learn more)
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