NuGEN’s Ovation RNA Amplification Systems are powered by Ribo-SPIA® technology

Ribo-SPIA® technology provides an elegant method for linear, isothermal amplification of the mRNA species in a total RNA population. Unlike exponential amplification, this linear amplification approach is carried out by replication of only the original transcripts not replication of copies, resulting in a high-fidelity, robust and sensitive amplification process. Amplification of the global mRNA population within total RNA using Ribo-SPIA® technology involves a simple three-step process that can be completed in less than four hours. This process has been employed in 3’-initiated amplification as well as in whole transcriptome (WT) approaches. View both processes in action by clicking on the Technology Animation links, or view the static graphic representation.

Process:
Step 1. First-strand cDNA is produced using a unique DNA/RNA chimeric primer mix (3’ or WT) and reverse transcriptase. The DNA portion of the primer contains either a poly T sequence which anneals to the 3' poly(A) sequence of each polyadenylated transcript or has a random sequence that will anneal to random sites across the whole transcriptome. The RNA portion of the primer contains a unique sequence used to incorporate a priming site for the linear amplification step (Step 3 below).

Step 2. Second-strand cDNA is synthesized with DNA polymerase. This cDNA strand has the same sense orientation as the original mRNA. The 3' end of the newly synthesized, second cDNA strand will contain the complement to the unique RNA sequence in the first-strand chimeric DNA/RNA primer.

Step 3. The SPIA linear amplification step uses a second DNA/RNA chimeric primer, DNA polymerase and RNase H in an isothermal reaction. RNase H removes the short RNA portion of the double-stranded cDNA revealing a site for binding the second DNA/RNA chimeric primer. DNA polymerase synthesizes cDNA starting at the 3´ end of the primer and displacing the existing forward strand. RNA at the 5´ end of the newly synthesized strand is again cleaved by RNase H partially exposing the priming site for initiation of the next round of DNA synthesis. The entire process is repeated continuously with multiple DNA polymerase molecules participating in DNA synthesis along the same template molecule, leading to the rapid accumulation of micrograms of amplified single-stranded antisense (opposite sense) cDNA products.

The Ovation® System family of products is covered by U.S. Patent Nos. 6,692,918, 6,251,639, 6,946,251 and 7,354,717, and other issued and pending patents in the US and other countries. NuGEN, Ovation, SPIA, Ribo-SPIA, WT-Ovation, FL-Ovation, and Imagine More From Less are registered trademarks or service marks of NuGEN Technologies, Inc. All other marks appearing in these materials are marks of their respective owners.