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. 2018 Aug 10;11:261. doi: 10.3389/fnmol.2018.00261

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Copyright © 2018 van den Hurk, Erwin, Yeo, Gage and Bardy.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Expression analysis of ERCC RNA spike-in controls to assess technical performance of SMARTer cDNA preparation. (A–C) Expression of a high-, medium-, and low-copy ERCC standard in n = 28 spiked cells measured by qRT-PCR and RNA-seq. Variability in expression increases with a decrease in absolute ERCC copy number. (D) Measured expression of ERCC RNA spike-ins as a function of the number of actual molecules added to each sample. The very small amount (∼10 pg) of RNA in a single cell requires a significant dilution of the ERCC stock to avoid overspiking of the experimental sample with exogenous transcripts. With a 1:5,000,000 dilution in the final master mix before reverse transcription, 73 ERCCs were present in at least one copy number, 71 of which were detected by RNA-seq across samples (n = 28). (E) The variation in measured ERCC expression levels is highest for low expressed (i.e., low abundant) ERCCs.