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Nuclease Protection Assays: The Basics

the single-stranded, antisense probe must typically be completely homologous to target RNA to prevent cleavage of the probe:target hybrid by nuclease. This means that partially related sequences (e.g., probe and target RNA from different species) usually cannot be used.


Steps Involved in Nuclease Protection Assays

RNA Isolation
There are a number of protocols, techniques and commercially available kits that can be used to isolate RNA for NPAs, i.e., most if not all RNA isolation methods are compatible with NPAs. RNA isolation techniques all share these common attributes:

  • Cellular lysis and membrane disruption
  • Inhibition of ribonuclease activity
  • Deproteinization
  • Recovery of intact RNA

Ambion provides several options for isolation of total RNA and mRNA that are compatible with a variety of cells and tissues, including bacteria, yeast, plant and animal. For a further discussion of RNA isolation options, see RNA Isolation: The Basics.


Probe Generation and Purification
The type of probe used (RNA vs. DNA) is dependent upon which nuclease is used in the digestion step. Ribonuclease protection assays (i.e.,

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Ideally suited for resolving DNA and RNA 10-1000 bases especially PCR products ranging from 100-1000 bp. Gelling temp. (4%) 27C Melting temperature 65C. EEO: 0.10 Rnase/Dnase: none detected
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