• qNPA™ Technology in Formalin Fixed Paraffin Embedded (FFPE) Tissue
• Array Plate Platform
• Why qNPA?
• miRNA

qNPA™ Technology in Formalin Fixed Paraffin Embedded (FFPE) Tissue

How does qNPA analyze gene expression in FFPE material without RNA extraction?
In most FFPE tissue specimens, the majority of RNA is cross-linked to cellular material, much of which is insoluble and is therefore resistant to RNA extraction. qNPA oligonucleotides can hybridize to these tissue-bound RNAs and the nuclease protection assay can proceed to completion with detection of both soluble and insoluble RNA.

How much FFPE material do I need to use per well?
Typically, a 1cm² portion of a 5µm tissue section is sufficient for a strong signal. However, the required amounts may vary by specimen and fixation types. Please contact HTG Molecular for more information.

Can I perform qNPA testing on slides that have already been stained?
We have preformed qNPA on both miRNA & mRNA over multiple slides with results demonstrating recovered genes of > 90%

How do I prepare the FFPE material?
Unlike most techniques which require tedious and expensive preparative steps, qNPA requires only that the section of fixed tissue be scraped off the glass slide and into a tube containing the HTG Molecular lysis buffer. The entire qNPA process is described here.

Do I need to remove the paraffin from my samples?
No. Any paraffin in the samples melts during incubation in the HTG lysis buffer and will float to the surface of the sample well. The oil overlay used to prevent evaporation during this step will effectively remove any molten paraffin. qNPA technology does not use xylene or other volatile organic solvents.

How does gene expression analysis by qNPA technology compare to IHC staining?
Depending on the intent of the test, qNPA often performs better and provides more data per sample than a single IHC slide. In this illustration, individual tissue slices were stained and examined for the presence of BCL-6. The signal obtained from qNPA (chart) correlates well with the IHC staining.

Are there references for the use of qNPA to analyze FFPE?
Please refer to our publications page for a comprehensive list of publications referencing qNPA technology.

Array Plate Platform

Is the array plate platform right for my research?
Any laboratory that is currently analyzing gene expression levels, especially those utilizing PCR, microarrays or similar technologies, can easily transition to HTG Molecular’s array plate and qNPA technology. It is ideal for labs that need multiplexed, high-throughput data, including pharma, and research labs. The same qNPA technology can be implemented in any of these three types of labs for a seamless transition between each stage of the process.

What sample types are compatible with qNPA technology?
qNPA technology provides accurate, reproducible results with a wide variety of sample types, including cultured cell lines, fresh tissue and FFPE tissue. For questions regarding a sample type, please contact us for more information.

Why qNPA?

qNPA technology offers numerous important advantages over other gene expression analysis techniques:

• No RNA extraction: Run samples from frozen tissue, FFPE, plants, and bacteria without RNA extraction. Not only does this remove a time-consuming and costly bottleneck in high throughput analysis, but it also reduces a potential source of technical sample variation.
• No cDNA synthesis: Traditional methods of gene expression analysis require a reverse transcription of the targeted RNA. qNPA-based analysis does not involve this step, thereby removing another source of potential sample variation.
• No RNA amplification or labeling: qNPA uses standard DNA oligonucleotides and S1 nuclease, greatly simplifying the experimental process. Coupled with cDNA synthesis, many analysis platforms require a time-consuming RNA amplification and biotin labeling step, adding more cost and potential sample variation.
• Up to 47 gene multiplexing: The qNPA array plate platform will simultaneously measure up to 47 genes in the same sample well. Other multiplexing platforms, such as qPCR, only reliably multiplex 4-6 genes per well, and require extensive development and verification.
• Consistent and reproducible data: Well-to-well CV’s are typically ~10%, far superior to qPCR or microarray data. Data is reliable and consistent, allowing different wells and plates to be compared with confidence and without complex normalization schemes.

miRNA

What advantages does the qNPA array plate have over other miRNA technologies?
No miRNA Extraction: HTG Molecular’s qNPA technology uses a simple lysis step to obtain the miRNA for analysis, not an expensive, time-consuming, and artifact-introducing miRNA extraction procedure. Not only does this remove a time-consuming and costly bottleneck in high-throughput analysis, but it also reduces a potential source of technical sample variation.

How can HTG Molecular detect both miRNA and mRNA analysis in the same sample well?
The qNPA portion of the protocol is identical for both miRNA and mRNA assays. The detection process for miRNA and mRNA is similar, but slight modification is required when the two are combined in the same well. Please contact HTG Molecular for more information.