Development of ERCC RNA Spike-In Control Mixes

The National Institute of Standards and Technology (NIST) hosted External RNA Control Consortium (ERCC) has been working since 2003 to generate a common set of RNA controls that can be used in gene expression measurements. These controls have been designed to mimic natural eukaryotic mRNA sequences with the ability to be used across multiple measurement platforms. These controls open-up the possibilities for technical evaluation of multiple gene expression systems including real-time quantitative PCR, one-color and two-color microarray systems, next generation sequencing platforms, as well as serve as an in-process quality control check for library amplification processes. The long standing history of Ambion® as the RNA Company creates a great opportunity to formulate the ERCCs controls into useful mixes that can be spiked into RNA after isolation. Each mix contains 92 of the ERCC controls in a mixture that spans over 6 logs o dynamic range. The 92 transcripts in each mix are further divided into 4 sub-pools that can be utilized to evaluate fold-change measurements of gene expression between the two mixes. TaqMan® Gene Expression assays targeting the 92 transcripts are utilized to monitor progress through the transcriptome library preparation steps required to create a next generation sequencing (NGS) library from RNA. We demonstrated the use of TaqMan® assays to inform a researcher of library quality prior to continuing with an expensive and time consuming next generation sequencing experiment. These controls are platform agnostic and provide informative data for multiple library generation methods targeting various gene expression measurement platforms from RNA. ERCC RNA spike-in control mixes from the NIST traceable ERCC plasmids has great potential in the hands of our customers to open-up new capabilities in understanding variability in RNA preparation, RNA library preparation, determining detection limits of measurement systems, and informing downstream analysis. As such, we report here the first in a potential line of RNA control products leveraging the certified plasmid sequences from NIST.