qNPA™ Overcomes Common FFPE Problems
qNPA™ Technology is uniquely suited to produce robust gene expression results from FFPE and other fixed tissues where RNA quality is an issue. These samples typically yield poor gene expression results due to RNA cross-linking and fragmentation. Due to the inaccessibility of the RNA, significant time and resources are required to prepare the samples.
For more technical information regarding qNPA-based gene expression in fixed tissues, read our FFPE FAQ or contact our technical service group.
1. Damaged or fragmented RNA
Problem: Causes small, incomplete cDNA transcripts to be formed. Most techniques will also suffer from a strong 3′ bias in the data analysis results.
qNPA Solution: qNPA does not require cDNA synthesis, and the qNPA oligonucleotides can be arranged throughout the transcript. If the RNA fragments are over ~75 bases, qNPA is unaffected by RNA fragmentation.
2. Soluble RNA with cellular material linked to it
Problem: Reverse transcriptase polymerases often cannot proceed due to physical blocking of the elongation process. This results in short, incomplete cDNAs.
qNPA Solution: As with degraded RNA, ‘dirty’ RNA with small amounts of cellular material attached to it can be measured thanks to by multiple probe placements throughout the transcript.
3. RNA bound to insoluble cellular material/low amounts of soluble RNA available for extraction
Problem: Much of the RNA in fixed tissue samples in bound to large pieces of cellular debris preventing it from being soluble and available for purification. Large amounts of precious FFPE tissues are wasted.
qNPA Solution: qNPA does not use an RNA extraction prior to analysis. qNPA oligos can enter cellular debris complexes, be protected by non-soluble, cross-linked RNA from S1 nuclease digestion, and collected for measurement on the ArrayPlate platform. Less sample is needed; none is wasted.
4. Contaminants and inhibitors in the ‘purified’ RNA
Problem: Common contaminants such as paraffin, melanin, and polysaccharides are often carried-over in RNA extraction techniques. These contaminants will inhibit polymerases and cause poor results to be obtained.
qNPA Solution: The qNPA process effectively removes any paraffin in a sample with an oil-overlay step. The technology does not use polymerases or other enzymes which may be inhibited by polysaccharides.
