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qNPA™ 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.

FFPE Nuclease Array

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 can access the bound 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 to 3 cm square 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.

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.

How does gene expression analysis by qNPA 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.

FFPE - BCL Array

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!