RPM Protocol

 

The RPM assay protocol (Figure 3) is similar to most other nucleic acid-based protocols, and contains several standard procedures. Beginning with a clinical or environmental specimen, the steps are:

 

• Extraction of total nucleic acids (TNA) using one of several commercially available methods

   

  Reverse Transcription to convert any viral RNA to cDNA using random primers

   

  PCR endpoint amplification of pathogen-specific gene sequences using locus-specific, multiplexed primers

   

  Sample pooling and purification combines the four multiplex reactions, removes any residual primers, enzymes, salts, dNTPs, and concentrates the amplified DNA

   

  Fragmentation and labeling prepares the amplified targets to be hybridized to the microarray

   

  Hybridization, staining and washing allows for the detection of the target sequences on the microarray

   

  Internal Controls monitor the efficiency of amplification and hybridization steps

 

 

 

 

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