Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

Assessing technical performance in differential gene expression experiments with external spike- in RNA control ratio mixtures



Sarah A. Munro, Steven P. Lund, Patrick S. Pine, Hans Binder, Djork-Arne Clevert, Ana Conesa, Joaquin Dopazo, Mario Fasold, Sepp Hochreiter, Huixao Hong, Nederah Jafari, David P. Kreil, Pawel P. Labaj, Yang Liao, Simon Lin, Christopher E. Mason, Javier Santoyo, Steven J. Schrodi, Leming Shi, Wei Shi, Gordon K. Smyth, Nancy Stralis-Pavese, Zhenqiang Su, Charles Wang, Jian Wang, Joshua Xu, Zhan Ye, Yong Yang, Ying Yu, Paul Zumbo, Marc L. Salit


There is a critical need for standard approaches to assess, report and compare the technical performance of genome-scale differential gene expression experiments. Here we assess technical performance with a proposed standard ‘dashboard’ of metrics derived from analysis of external spike-in RNA control ratio mixtures. These control ratio mixtures with defined abundance ratios enable assessment of diagnostic performance of differentially expressed transcript lists, limit of detection of ratio (LODR) estimates and expression ratio variability and measurement bias. The performance metrics suite is applicable to analysis of a typical experiment, and here we also apply these metrics to evaluate technical performance among laboratories. An interlaboratory study using identical samples shared among 12 laboratories with three different measurement processes demonstrates generally consistent diagnostic power across 11 laboratories. Ratio measurement variability and bias are also comparable among laboratories for the same measurement process. We observe different biases for measurement processes using different mRNA-enrichment protocols.
Nature Communications


RNA, gene expression, sequencing, interlaboratory, spike-in controls, ratios, RNA-Seq


Munro, S. , Lund, S. , Pine, P. , Binder, H. , Clevert, D. , Conesa, A. , Dopazo, J. , Fasold, M. , Hochreiter, S. , Hong, H. , Jafari, N. , Kreil, D. , Labaj, P. , Liao, Y. , Lin, S. , Mason, C. , Santoyo, J. , Schrodi, S. , Shi, L. , Shi, W. , Smyth, G. , Stralis-Pavese, N. , Su, Z. , Wang, C. , Wang, J. , Xu, J. , Ye, Z. , Yang, Y. , Yu, Y. , Zumbo, P. and Salit, M. (2014), Assessing technical performance in differential gene expression experiments with external spike- in RNA control ratio mixtures, Nature Communications, [online], (Accessed April 15, 2024)
Created September 25, 2014, Updated November 10, 2018