qNPA™ Technology in Fixed Tissues (FFPE)
How does qNPA analyze gene expression in FFPE material without RNA extraction?
In most fixed 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 as normal.
As the image to the left shows, a significantly larger amount of signal is obtained from the FFPE samples (column A) which include the pellet material in the qNPA process. This is distinctly different from the fresh tissue control (column B), where the material is fresh frozen and RNA is readily soluble in the supernatent. Only qNPA technology can access the bound, cross-linked RNA in FFPE that RNA extraction techniques cannot recover, providing an advantage over other gene expression techniques.
How much FFPE material do I need to use per well?
It depends on the sample, the transcripts being measured, and the cell density, but typically a 2 X 3 cm portion of a 5µm tissue section is sufficient for strong signal. More tissue may be needed if samples are to be run in duplicate or triplicate. Sample requirements for other fixed cell preparations may vary, contact us for more information.
Can I do qNPA testing on slides that have already been stained?
It depends on the stain and the protocol. H & E stained slides have been used to obtain gene expression results identical to those of matched unstained slides. Other stains and staining protocols may be acceptable if they do not damage the RNA through high temperatures or metal ion deposition. HTG can test your stained slides to see if they work with qNPA technology.
How do I prepare the FFPE material?
Unlike most techniques which require tedious and expensive preparative steps, qNPA requires the researcher only to scrape the section of fixed tissue off the glass slide and into a tube containing the qNPA Lysis Buffer. The sample is then overlain with oil, heated, and the qNPA protocol proceeds as outlined here.
Do I need to remove the paraffin from my samples?
No. Any paraffin in the samples is melted during incubation in our 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 does not use xylene or other volatile organic solvents.
Does HTG provide CLIA testing services?
HTG has recently partnered with Clinical Reference Laboratory of Lenexa, Kansas to provide CLIA certified testing with qNPA products. Please contact your local HTG sales representative for further information.
How does gene expression analysis by qNPA technology compare to IHC staining?
Very well. 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?
Roberts, R., Sabalos, C., Martel, R., et al, ( 2007). “Quantitative Nuclease Protection Assay in Paraffin-Embedded Tissue Replicates Prognostic Microarray Gene Expression in Diffuse Large-B-Cell Lymphoma” Laboratory Investigation, 87: 979-997.
Rimsza, L., LeBlanc, M., Unger, J., et al., (2008). “Gene expression predicts overall survival in paraffin embedded tissues of diffuse large B cell lymphoma treated with R-CHOP” Blood, Online June 10, 2008 doi: 10.1182/blood-2008-02-137372.
Please contact our technical support with any further questions!
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.
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.
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.
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?
We can detect any of the human, rat and mouse miRNAs listed in the Sanger Institute database. 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!
How do I get started?
I’m interested in the qNPA ArrayPlate, what format does it come in?
HTG currently offers the ArrayPlate in two different formats:
Our qFixed Arrays each contain 16 genes focused on a particular functional area. These assays come in a ready to run, preconfigured format. Only a simple ‘tuning’ step is required prior to running the assay. This tuning step can be performed as part experiment is samples are small and precious.
The qCustom system allows researchers to develop their own custom arrays with the gene content of their choice. These arrays are configured and QC’d by our in-house development team to ensure the assay does not contain cross-reactive array elements.
Either ArrayPlate product can be used in your lab (with our imagers) or run by our skilled team at our facility in Tucson, Arizona.
I want to design a qCustom Array. What does HTG need from me?
To begin the design process for your qCustom array, our scientists will need three items:
- A gene list of up to 46 genes that you want represented on your array.
- A lysate sample representing the ‘uninduced’ or basal state of your gene expression targets
- A lysate represented the expected ‘full induction’ of your specified genes
The lysates are used to tune the qCustom array for maximum data coverage for your specific sample. Our experienced scientists will help you determine the appropriate conditions for creating these lysates. Once the array is tuned, it is ready to be used in either your lab or in our service facility.
I am interested in using a qFix™ ArrayPlate. What do I need?
To used our qFixed Arrays in your own lab, you will need to have a high resolution luminescence imager; HTG offers two imagers for this purpose. Other imagers may be suitable, please contact us for more information.
Other equipment that is strongly suggested includes robotic liquid handling systems and plate washers. Our scientists have found that, although the assay can be performed with standard or multi-channel pipettes, the reproducibility of the assay improves significantly when automation is used.
What do I need to do to use HTG’s services?
Most of our service customers prepare their cells or other samples (such as tissue or FFPE preparations) in their own facilities using their own specifications. When these treatments are finished, they add qNPA Lysis Buffer (supplied by HTG) to the wells to kill the cells and release the RNA. These lysates are frozen and shipped to HTG on dry ice for immediate processing. We typically deliver the final data set from plates we receive in less than 10 business days.
How much do HTG’s products and services cost?
Please contact your regional sales representative for pricing information. Due to the nature of their projects, many of our customers receive volume discounts. We want to be able to provide you with an accurate estimate of your project costs.
Our pilot studies are an ideal way to familiarize yourself with the power and benefits of the ArrayPlate Technology. Contact us for more information regarding setting a pilot study up for you.
Is the ArrayPlate platform right for my research?
Any researcher who is looking to analyze gene expression in a large number of samples for specific gene targets should benefit from using the qNPA™ ArrayPlate Platform. The ArrayPlate may be especially useful in your research if you are:
- Using large amounts of multiplexed qRT-PCR
- Performing TaqMan® or similar experiments on multiple genes
- Analyzing many samples with microarrays
The ArrayPlate platform is designed to be a high throughput assay performed with robotic liquid handling equipment. It is ideally suited to analyze gene targets identified and validated using lower throughput systems. We have found that most of our customers:
- Have used microarrays to identify their initial genes of interest
- Examine a relatively small number of genes to analyze per follow-up sample
- Intend to examine at least 100 samples per experiment
They have turned to using the ArrayPlate Platform due to its fast and simple protocol, low cost per data point, and highly reliable data. Customers with other research needs may benefit from our ArrayPlate or qNPA technologies as well. Please contact your regional account representative to find out if HTG has a solution for your research goals.
Sample Types used with the qNPA™ ArrayPlate™ Assay
The qNPA™ ArrayPlate™ Assay has been used with a wide variety of sample types, including cultured cell lines, fresh tissue pieces, and FFPE archives. For questions regarding your sample type, please contact us for more information.
Immortalized Cell Lines
Extracted RNA
FFPE and H & E Stained Slides
Primary Cell Lines and Tissues
adrenals lung
bladder lymphocytes
blood muscle
brain ovaries
breast pancreas
colon prostate
ear punches spleen
heart testes
kidney umbilical cord
liver
Plants
Maize
Rice
Whole Organisms
Arabidopsis
Drosophila
Gram negative bacteria
Gram positive bacteria
Why qNPA™ ?
Quantitative Nuclease Protection Assay (qNPA™)
qNPA Technology offers several important advantages over other gene expression analysis techniques:
No RNA extraction: HTG has successfully run samples from cultured cell lines, primary cells, tissue pieces, FFPE, plants, and bacteria without RNA extraction (For a complete list, click here). 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: Coupled with cDNA synthesis, many analysis platforms require a time-consuming RNA amplification and biotin labeling step, adding even more cost and potential sample variation. qNPA uses standard DNA oligonucleotides and S1 nuclease, greatly simplifying the experimental process.
Sensitivity: Single-gene copy sensitivity in as few as 1,000 cells – Measure as few as 1,000 molecules of RNA per well with CV’s of ~10%.
True 47 gene multiplexing: The qNPA ArrayPlate 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.
Differential expression resolution:qNPA can regularly and accurately detect gene expression changes of as little as 20% (or 1.2 fold) in a plate that has a CV of 10%. qPCR provides reliable resolution of only 50% (or 1.5 fold), which can miss biologically significant changes.
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