The Ambion® kit contains reagents for performing ribonuclease protection assays, an extremely sensitive technique for the detection, quantitation, and characterization of RNA. Sufficient reagents are included for 100 reactions.
• Ideal for new users
• Simplified single-tube reaction
• Sensitive—detect as little as 5 fg of target mRNA
• No phenol extraction or Proteinase K digestion
• Ideal for multiprobe –analysis—up to 12 genes analyzed simultaneously in each sample
• Used for detection, quantitation, and mapping of mRNA
Fast, Sensitive Assay
The procedure for the RPA III™ Kit is fast and takes place in a single microfuge tube. Hybridization occurs in solution rather than on a solid support (i.e., nylon membrane), allowing more complete hybridization of the antisense probe to the target mRNA. After hybridization, excess single-stranded probe and unhybridized sample RNA are removed by digestion with a mixture of ribonucleases. Then, in a single step, ribonucleases are inactivated, and the protected double-stranded RNA (probe + target) is precipitated. The products are separated on a denaturing polyacrylamide gel and then visualized by autoradiography when isotopic probes are used or transferred to a membrane for nonisotopic detection protocols.
Use Multiple Probes with a Single RNA Sample
In current research facilities, it is rarely sufficient to assess the expression level of a single gene. Ideally, multiple targets and one or two internal controls would be assessed within individual samples. For such analyses, nuclease protection assays are without peer among the commonly employed methods of RNA analysis. Five to ten probes plus one or two internal controls can routinely be used in a single assay, allowing simultaneous quantitation of multiple messages within a single RNA sample.
Additional Benefits of RPAs
Northern blots are limited by the amount of total RNA that can be loaded into a single lane without overloading the gel (typically ~20 µg per lane). With ribonuclease protection assays as much as 100 µg of RNA may be used since hybridization takes place in solution, and the nonhybridized RNA will be degraded before gel analysis. Additionally, because probes for protection assays are significantly shorter than the mRNA species being detected, the target RNA need not be completely intact (breaks that occur outside the region that hybridizes to the probe will not affect RPA data but will result in smeared bands on northerns).