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. 2019 Mar 20;9:4836. doi: 10.1038/s41598-019-41235-9

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Optimized ERCC spike-in dilutions assess poly(A) PCR sensitivity and dynamic range without suppressing cDNA amplification of endogenous transcripts. (A) 100 pg RNA was supplemented with ERCC Mix 1 at the indicated dilutions and amplified via optimized poly(A) PCR. ERCC and endogenous gene abundances were measured by qPCR, and data are shown in grayscale as the inverse quantification cycle (40–Cq) from n = 4 amplification replicates. Negative effects of the ERCC spike-ins on endogenous genes (lower) were assess by two-way ANOVA with replication. (B) ERCC Mix 1 (6.23 × 10⁴ copies) was spiked into 100 pg RNA and amplified via optimized poly (A) PCR. Proportional abundance of ERCC standards was estimated with a seven-log dilution series from purified qPCR end products. Data are shown as the median 40–Cq (black) for 22 ERCC spike-in standards from n = 8 amplification replicates (gray) with undetected “dropouts” reported below (circles).