| 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 dependant upon which nuclease is used in the digestion step. Ribonuclease protection assays (i.e., RPA III™, RPA II™, HybSpeed™ RPA, and Direct Protect™ Lysate RPA Kits) all require the use of RNA probes. It is essential that probes used in NPA analysis are all of a discrete length. High specific activity single-stranded RNA probes can be produced by in vitro transcription reactions using Ambion's MAXIscript™ Kit. To assure that the probe is full length we recommend gel purification. Ambion's Technical Bulletin 171 discusses gel purification of probes in detail. Either radiolabeled or nonisotopically labeled probes can be used for NPA analysis. We recommend that nonisotopic nucleotides be incorporated enzymatically (versus post synthesis chemical labeling of probes). Technical Bulletin 173, "Methods for Nonisotopic Labeling," describes how the MAXIscript Kit can be used to incorporate nonisotopic nucleotides into RNA probes. For accurate quantitation of a specific message, probe concentration must be in molar excess over the target mRNA. This necessitates the use of low specific activity probes for abundant targets. For moderately abundant messages (e.g., ß-actin, GAPDH, or cyclophilin), a 1:50 dilution of labeled NTP with "cold" NTP should be used to increase the molar amount of probe made while simultaneously reducing the specific activity of the probe. For very abundant messages (e.g., 18S rRNA or 28S rRNA), a 1:10,000 dilution with "cold" NTP should be used. NPA Analysis
Ambion's NPA kits provide many advantages over traditional and homebrewed NPAs. These advantages include: - Ease of use. A patented technology is used which allows for a single-tube reaction with no phenol extraction or proteinase K digestion.
- Increased sensitivity. Ambion's RPA III™ Kit is the most sensitive RPA available.
- Faster reactions. Ambion's HybSpeed™ RPA Kit has a revolutionary 10-minute hybridization step allowing an RPA reaction to be completed in a single day.
Denaturing Acrylamide Gel Electrophoresis
The number and size of probes will dictate the gel size and acrylamide concentration. Typically a 5% denaturing acrylamide gel is used. This will effectively resolve fragments of about 500–1000 nucleotides. It is useful to have size markers on the gel. Single-stranded RNA markers are the most accurate (e.g., Ambion's RNA Century Markers or Century Marker Templates), but double-stranded DNA markers can be used if it is not critical to know the exact size of the products. |