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miRNA on the qNPA™ ArrayPlate

What advantages does the qNPA ArrayPlate have over other miRNA technologies?

No RNA Extraction

qNPA based technologies use a simple lysis step to obtain the RNA for analysis, not an expensive, time-consuming, and artifact-introducing RNA extraction procedure. This not only simplifies the workflow of gene expression analysis, but it also reduces the amount of noise introduced in to the data measurements. qNPA measures the changes in gene expression due to biology, not the choice of RNA extraction technique.

Sensitivity

The qNPA ArrayPlate platform will routinely detect as few as 15,000 miRNA molecules in a sample well. With a typical sample size of 15 to 25 thousand cells, we can reliably detect a miRNA present at one transcript per cell. The no-amplification nature of the qNPA System ensures that signal is not obtained due to amplification artifacts.

Selectivity

Spurious signal due to cross-hybridization of closely related sequences is minimal in the ArrayPlate, unlike other technologies.

miRNA Sequence

Two closely related miRNAs were tested on the qNPA ArrayPlate and a leading miRNA RT-qPCR detection technology. The same amount of RNA oligonucleotides were added to each sample.

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miRNA Chart 2

When matched with the gene specific probe set (data in the blue boxes), the RT-qPCR technique produced CT values well within the expected range. The experiments where the RNA substrate and the probe sets were mismatched (data in the white boxes), however, also regularly produced CT values well within the range typically regarded as reliable data. The significant amount of cross reactivity in these reagent sets indicates they provide incorrect, unreliable data, and are not suitable for research situations where this level of discrimination are needed.

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miRNA Chart 1

When the same RNA oligonucleotides  were applied to the qNPA ArrayPlate, very minimal signals were obtained from the mismatched spots (data in the white boxes), indicating good discrimination of the closely related miRNA sequences.

How can HTG detect both miRNA and mRNA analysis in the same sample well?

The qNPA portion of the protocol remains the same when miRNAs are targeted. Detection by the ArrayPlate requires a small protocol change to allow for Avidin-HRP binding, while data acquisition and analysis is performed as with the normal ArrayPlate.

Other expression technologies require significantly different reverse transcription, labeling, or detection schemes to detect miRNAs, and these technical approaches often cannot be applied to mRNAs in general. Normalizing expression data becomes difficult because the standard mRNA housekeeping genes cannot be detected on the same platform. Often the results of two different detection technologies need to be fitted together to create a picture of miRNA expression levels, requiring statistical interpretation and less reliable results.

How is detecting miRNAs different from detecting normal mRNA transcripts with the qNPA ArrayPlate?

miRNAs are significantly shorter (typically 21 to 23 bases) than mRNA and cannot protect the standard 50 nucleotide-long DNA oligo used for mRNA detection during the S1 nuclease detection step. Therefore, we have designed smaller qNPA protection oligos to detect these miRNA sequences.

These miRNA-targeted qNPA protection oligos cannot support hybridization of the Detection Linker oligonucleotide due to their short length. Therefore, these qNPA oligonucletides are biotinylated to facilitate subsequent detection. An avidin-HRP conjugate is used to detect miRNA hybridization instead of the Universal Detection Linker used in the standard protocol.

What percentage of known miRNAs can be measured with the qNPA ArrayPlate?

Currently ~97% of miRNA sequences in the Sanger Institute database can be detected using our approach. A small percentage of miRNAs with very low melting temperatures ( A/T rich sequences) are not currently compatible with our approach. Please contact us for more details.

Can the ArrayPlate detect other small RNAs?

Yes. The technology behind miRNA detection in the qNPA ArrayPlate can be used to detect other small RNA molecules that are normally present in the cell or introduced via transformation or transfection techniques.

Have more questions about the qNPA ArrayPlate? Contact our technical services!