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.
