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Novel small RNA spike-in oligonucleotides enable absolute normalization of small RNA-Seq data


Normalization of high-throughput small RNA sequencing (sRNA-Seq) data is required to compare sRNA levels across different samples. Commonly used relative normalization approaches can cause erroneous conclusions due to fluctuating small RNA populations between tissues. We developed a set of sRNA spike-in oligonucleotides (sRNA spike-ins) that enable absolute normalization of sRNA-Seq data across independent experiments, as well as the genome-wide estimation of sRNA:mRNA stoichiometries when used together with mRNA spike-in oligonucleotides.


Data from sRNA-Seq experiments are typically normalized and reported in relative terms such as reads per million genome-matching reads (RPMs)1. Relative normalization works well if it is assumed that the sRNA sub-populations have equal proportions across the different tissue types being profiled. However, this assumption is often invalid because sRNA populations are frequently dynamic across different tissue types and in various mutant backgrounds2,3,4,5,6,7. Therefore, the standard practice of comparing relatively normalized sRNA-Seq values can produce misleading results. In contrast, absolute normalization of sRNA-Seq data should enable accurate comparisons of small RNA levels in different cell types, mutant tissues or disease states on a genome-wide scale. However, previous attempts to use exogenous sRNA oligonucleotides for absolute normalization of sRNA-Seq data had variable success8,9,10 and thus have not been widely used. The varying sequencing efficiencies of specific sRNA spike-ins are most likely due to their inherent properties, such as variable secondary structures, that influence their ligation efficiency during sRNA-Seq library construction11,12,13. For example, previously an additional correction factor was required to scale individual sRNA spike-in amounts to account for the non-linear relationships between the molar amount of exogenous sRNA spike-ins added to a sample and the corresponding number of reads sequenced10. Here we present a novel sRNA spike-in oligonucleotide design and demonstrate how sRNA spike-ins can be used for robust absolute normalization of sRNA-Seq data across independent experiments. Moreover, we demonstrate how sRNA spike-ins can be used in combination with commercially available mRNA spike-in oligonucleotide mixes to compare values generated by sRNA-Seq and mRNA-Seq.


To generate a set of exogenous sRNA spike-ins for absolute normalization of sRNA-Seq data, we designed 21 nucleotide (nt) long RNA oligonucleotides with three main features (Fig. 1a). First, the sRNA spike-ins contained a 5′ monophosphate and 2′-O-methyl group in order to mimic endogenous plant small RNAs. The 2′-O-methyl group is common to plant small RNAs14, but this modification could be omitted if investigating animal miRNAs for instance. Second, the sRNA spike-ins contained a semi-random 13 nt core sequence that does not match the genome-of-interest (e.g. in this study Arabidopsis thaliana). Third, these semi-random 13 nt core sequences are flanked by a set of four randomized nucleotides on both the 5′ and 3′ ends. Eight sRNA spike-ins with different 13 nt core sequences were designed and mixed in specific molar ratios as shown in Fig. 1a. These were added to total RNA prior to sRNA-Seq library preparation. After sequencing, the non-genome matching 13 nt unique tags were used to quantify reads derived specifically from each sRNA spike-in. Each of these 13 nt core sequences can be represented by up to 65,536 possible 21 nt sequences. Because individual sRNA sequences have variable properties, such as secondary structure, that bias their representation in the final sRNA-Seq libraries11,12,13, the large number of diverse sequences that can be assigned to each 13 nt core sequence of the sRNA spike-in is expected to minimize cloning biases of each spike-in set as a whole. Accordingly, the sRNA spike-ins enabled the robust generation of standard curves for absolute data normalization in terms of molecules detected per µg total RNA (MPU) (Fig. 1b). Nearly perfect positive correlations (all had Pearson’s r values ≥ 0.99 and P < 7.42 × 10−6) were observed when plotting relative RPM levels reported by sRNA-Seq and the known absolute MPU amounts of sRNA spike-ins added to each sample (Fig. 1b and Supplementary Fig. 1a–e). As a proof of concept, the highly linear relationships between relative and absolute values were used to generate linear models to predict the number of miRNA molecules per µg of total RNA isolated from wild-type (Col-0) flowers (Fig. 1c).

Figure 1

Small RNA spike-in design and use as a tool to estimate absolute small RNA levels. (a) Design of small RNA spike-in oligonucleotides. Key features of small RNA spike-ins are shown in different colors corresponding to the key. Molar amounts of oligonucleotides added per µg of total RNA are indicated in parentheses. (b) Scatter plot of relative small RNA spike-in levels (reads per million genome-matching reads) compared to absolute small RNA levels (molecules detected per µg of total RNA) in Col-0 flowers (biological replicate 1). Pearson’s r value is indicated, as well as a dashed line that represents a linear model derived from the plotted values. (c) Density plot of individual miRNA family levels in Col-0 flowers (biological replicate 1) in molecules detected per µg of total RNA. Vertical dashed line indicates the median number of molecules per miRNA family.

Because the sRNA spike-ins enabled accurate data normalization (Fig. 1 and Supplementary Fig. 1a–e), we compared sRNA sub-populations in relative RPM and absolute MPU terms to determine if the two normalization procedures yield different results. Although similar analyses could be performed on any species, we used Arabidopsis thaliana to test the utility of the sRNA spike-ins because the Arabidopsis thaliana genome has well-annotated sRNAs and transcript models, different tissue types are easy to obtain and mutants deficient in major sRNA sub-populations are viable with only minor morphological effects. Plant sRNA populations consist of four main classes: 20–22 nt microRNAs (miRNAs) and trans-acting siRNAs (tasiRNAs) that tend to post-transcriptionally regulate protein-coding genes, and 20–22 nt and 23–24 nt small interfering RNAs (siRNAs) that typically arise from and silence transposons15. The proportions of sRNA sub-populations vary between flowers and leaves (Supplementary Fig. 2), which makes comparisons of relatively normalized leaf and flower sRNA levels prone to errors. For example, 30% and 53% of the respective Col-0 flower and Col-0 leaf sRNA populations were composed of 20–22 nt sequences (Supplementary Fig. 2a–c), but it cannot be determined whether there are absolutely more 20–22 nt sRNAs in leaves or whether this relative increase is merely due to reduced 23–24 nt siRNA levels in leaves compared to flower tissues. Using sRNA spike-ins, we compared relative and absolute normalization methods on sRNA sub-populations in Col-0 flowers and leaves. The two normalization methods produced different results. For example, whereas total miRNA levels have a significantly higher RPM in Col-0 leaves compared to Col-0 flowers (1.7-fold; P = 3.2 × 10−3; two-sample Student’s t-test), the absolute number of miRNAs are non-significantly reduced 1.5-fold in Col-0 leaves compared to Col-0 flowers (P = 0.13; two-sample Student’s t-test) (Supplementary Fig. 2d,e). Moreover, absolute, but not relative, normalization shows that miRNA families have significantly increased levels in Col-0 flowers compared to Col-0 leaves (Fig. 2a,b) (P = 4.1 × 10−3; two-sample Kolmogorov-Smirnov test). Therefore, relative normalization of sRNA-Seq data followed by comparisons between tissue types can lead to misleading results, which are mitigated through the use of sRNA spike-ins.

Figure 2

Small RNA spike-ins enable accurate comparisons of small RNA levels. (a and b) Violin plots of individual miRNAs, tasiRNAs and siRNA family levels in either relative (a) or absolute (b) units. P-values were based on two-sample Kolmogorov-Smirnov tests. P < 0.05, P < 0.01 and P < 0.001 are indicated by*, ** and ***, respectively. Violin plots are based on the mean among two bioreplicates for 93 miRNA, 14 tasiRNA, 6,361 20–22 nt siRNA and 5,952 23–24 nt siRNA families with ≥1 RPM in each bioreplicate. The 25th and 75th percentiles are indicated by the bottom and top of the vertical black bar in the violin plots, while the medians are indicated by white points within the vertical black bars. The top and bottom whiskers extend from the vertical black bar to the most extreme values within 1.5 times the interquartile range. The widths of the violins are proportional to the sample densities. (c and d) Scatter plots of the mean miRNA family levels among both bioreplicates for Col-0 flowers vs. Col-0 leaves (left) and Col-0 flowers vs. dcl234 leaves (right) in either relative RPM (c) or absolute MPU (d) terms. Red and blue points indicate miRNA families with significantly increased and significantly decreased levels, respectively. Significantly different miRNA levels were defined as having ≥2-fold difference and a p-value < 0.05 based on a two-sample Student’s t-test.

To further test how changes in sRNA populations can affect relative normalization, we generated sRNA-Seq libraries from siRNA-deficient flowers and compared these to the Col-0 flower datasets. More specifically, we generated sRNA-Seq libraries from flowers with null mutations in three genes encoding the DICER-LIKE2 (DCL2), DCL3 and DCL4 ribonucleases (i.e dcl234 flowers). DCL3 and DCL4 are required for 23–24 nt siRNA and tasiRNA biogenesis, respectively16,17,18,19,20 (Supplementary Fig. 2c). Therefore, dcl234 flowers are expected to have reduced tasiRNA and 23–24 nt siRNA levels. Comparisons of relative and absolute normalization methods on 20–24 nt siRNA levels produced similar results (Supplementary Fig. 2d,e and Fig. 2a,b). However, relative RPM levels for total tasiRNAs were not significantly reduced in dcl234 flowers compared to Col-0 flowers; whereas, absolute MPUs for total tasiRNAs were significantly reduced (P RPM = 0.08, P MPU = 0.04; two-sample Student’s t-test) (Supplementary Fig. 2d,e). Both relatively and absolutely normalized tasiRNA family levels are significantly reduced in dcl234 flowers but the decrease is more pronounced when using absolute terms (P RPM = 1.0 × 10−3, P MPU = 1.6 × 10−4; two-sample Kolmogorov-Smirnov test) (Fig. 2a,b). We also found that the total amount of miRNAs had both significantly higher RPMs and MPUs in dcl234 flowers compared to Col-0 flowers (P RPM = 0.04, P MPU = 0.02; two-sample Student’s t-tests) (Supplementary Fig. 2d,e). Therefore, in the absence of siRNAs, miRNAs are increased as has been previously proposed21.

The levels of individual sRNAs are often compared across sRNA-Seq datasets from various tissue types in order to determine when and where a particular sRNA is most abundant. We compared relatively and absolutely normalized values for 93 miRNA families in Col-0 flowers versus Col-0 leaves or dcl234 flowers to determine if the two normalization methods yield different results. Indeed, comparisons of RPM-based values suggest that 13 and 6 miRNA families have respectively increased and decreased levels in Col-0 leaves compared to Col-0 flowers (Fig. 2c). In contrast, due to the higher absolute miRNA levels in flowers (Supplementary Figure 2e and Fig. 2b), comparisons of MPU-based values indicate that no miRNA families are increased in Col-0 leaves compared to flowers, and 19 miRNA families are decreased in Col-0 leaves (Fig. 2d). Moreover, 35 miRNA families have increased levels in dcl234 versus Col-0 flowers based on RPM values; whereas, only 16 miRNA families are increased in dcl234 flowers compared to Col-0 flowers when using MPU values for the comparisons (Fig. 2c,d). Based on our results, absolute normalization of sRNA-Seq data from tissues with different underlying small RNA populations enables more accurate comparisons of both total and individual sRNA levels.

The relationships between sRNA levels and the abundance of either their precursors or targets are important to understand miRNA biogenesis and function. Because small RNAs and their longer RNA precursors/targets require different procedures for selection and cloning into RNA-Seq libraries, it is impossible to use relative normalization approaches to estimate the stoichiometries between sRNAs and their precursors or targets. We tested whether sRNA spike-ins together with commercially available mRNA spike-ins (ERCC spike-in mixes; LifeTech) enable cross-comparisons between sRNA-Seq and mRNA-Seq datasets. More specifically, we generated mRNA-Seq datasets from the same total RNA samples used to generate the sRNA-Seq data described above, and added ERCC spike-in mixes to the total RNA prior to mRNA-Seq library construction. Strong positive correlations between relative numbers of ERCC transcripts (transcripts per kilobase million; TPM) and the known number of molecules added per µg of total RNA were observed for all six datasets having Pearson’s r values of at least 0.96 and P  < 1.44 × 10−12 (Fig. 3a and Supplementary Fig. 3a–e). We then used these relationships to generate linear models and applied these to mRNAs with TPMs ≥ 1.0 in order to estimate mRNA MPUs genome-wide in all six datasets. This allowed us to then compare mature sRNA MPUs with those from either their precursors or targets. We found that miRNA/precursor ratios were similar in Col-0 leaves, Col-0 flowers and dcl234 flowers with respective median miRNA/precursor values of 2.1, 1.6 and 1.5 (Fig. 3b). The ratios between tasiRNAs and their precursors were higher, but not significantly different, than miRNA/precursor ratios in Col-0 leaves and Col-0 flowers with median tasiRNA/precursor ratios of 3.7 and 4.4, respectively (Fig. 3b). In contrast, tasiRNA precursors were more abundant than mature tasiRNA levels in dcl234 flowers with a median of 42.4-fold more precursors than tasiRNAs (Fig. 3b). This is consistent with the known requirement of DCL4 for tasiRNA biogenesis and the corresponding increase in tasiRNA precursor levels in dcl234 tissues16,17,18, 20, 22.

Figure 3

Combined use of small RNA and poly(A) RNA spike-ins allow direct comparisons of small RNA-Seq and mRNA-Seq data. (a) Scatter plot of relative (transcripts per million) and absolute (molecules per µg total RNA) ERCC poly(A) spike-in (LifeTech) levels. Pearson’s r value is indicated, as well as a dashed line that represents a linear model derived from the plotted values. (b) One-dimensional scatter plots of miRNA/miRNA precursor and tasiRNA/tasiRNA precursor levels in Col-0 leaves, Col-0 flowers and dcl234 flowers. Horizontal black bars represent the median of each population. (c) Violin plots of miRNA/target and tasiRNA/target levels in Col-0 leaves, Col-0 flowers and dcl234 flowers. Violin plots are as described in the Fig. 2 legend. P < 0.01 and P < 0.001 are indicated by ** and ***, respectively, and were calculated with two-sample Kolmogorov-Smirnov tests. The number (n) of miRNA:target and tasiRNA:target interactions examined is indicated.

We then investigated the stoichiometries between miRNAs and their targets genome-wide. We used publically available datasets of sRNA cleavage products (i.e. degradome datasets) from Col-0 flowers to select miRNA and tasiRNA targets23, 24. Ratios between miRNAs and their targets were not significantly different in Col-0 and dcl234 flowers and had median miRNA/target values of 3.9 and 0.63, respectively. In contrast, tasiRNA/target ratios were significantly different in Col-0 and dcl234 flowers as expected (P = 5.0e−16; two-sample Kolmogorov-Smirnov test) with respective 1.5-fold and 210.8-fold more target MPUs compared to tasiRNA MPUs (Fig. 3c). Therefore, in combination with mRNA spike-ins, sRNA spike-ins allow genome-wide estimations of precursor:sRNA and sRNA:target stoichiometries.


Small RNA spike-ins not only serve as useful internal controls for sRNA-Seq experiments requiring only 1–2% of the alignable reads for subsequent analyses, but can also be used to normalize sRNA-Seq data from different treatments, tissue types or research groups. The absolute normalization of sRNA molecules can be essential for accurate comparisons. Moreover, sRNA spike-ins enable genome-wide estimations of precursor:sRNA and sRNA:target stoichiometries, which are important for understanding these relationships on a molecular scale. Lastly, sRNA spike-ins could be used to assess cloning biases that exist in various sRNA-Seq library generation protocols for further improvements of such methods.


Plant material

The dcl234 mutants were composed of dcl2-1, dcl3-1 and dcl4-2t alleles as previously described17. Plants were grown in a controlled growth chamber at 20 °C–22 °C with a 16-h light/8-h dark cycle. Col-0 leaf samples were from rosette and cauline leaves isolated from 4–6 week old plants. Unopened floral buds were either collected from the same plants as the leaf samples (Col-0 flower) or from dcl234 plants (dcl234 flowers) grown under identical conditions.

sRNA spike-in design

An in-house script ( was used to generate a matrix consisting of the proportions of base identities for miRNA positions 5–17 (counting from the 5′ end of the miRNA) of the top 50% most highly abundant miRNAs and to semi-randomly select 1000 13 nt sequences. Two hundred and fifty-two sequences that did not perfectly align to the Arabidopsis thaliana Col-0 genome were considered further and all 256 possible 4-base combinations were added to both the 5′ and 3′ ends making 65,536 total sequences per set. The minimum free energies of endogenous miRNAs and all 21 nt RNAs within the 252 sets of 65,536 sequences were determined using RNAfold25 with the options –T 4 –noPS with in-house scripts ( and (, respectively. Minimum free energy distributions of endogenous miRNA and sRNA spike-in secondary structures were then examined with ( and eight sets of 21 nt RNA sequences with distributions similar to annotated miRNAs were selected for synthesis and ordered from Integrated DNA Technologies (IDT) (Supplementary Figure 4). The mix ratios were formulated to span the dynamic range of annotated Arabidopsis miRNAs.

RNA sequencing

Total RNA was isolated using TRIzol reagent (Thermo Fisher). The sRNA spike-in mix shown in Fig. 1a was diluted two-fold and added to 500 ng of total RNA prior to polyacrylamide gel size-selection of 18–75 nt RNAs followed by sRNA cloning using the NEBnext small RNA library prep kit for Illumina (NEB). ERCC spike-in mixes (LifeTech) were diluted 200-fold and 1 µl was added to 500 ng of total RNA. Ten ng of total RNA was used to generate mRNA-Seq libraries as described by Picelli et al.26. Samples were sequenced on an Illumina Hi-Seq 2500 sequencing machine in either single read 50 bp (sRNA-Seq) or paired-end read 50 bp (mRNA-Seq) modes. RNA-Seq datasets have been deposited into NCBI GEO (GSE98553).

Data analysis

Adaptor sequences were removed from sRNA-Seq reads with ( and the resulting FASTQ file was condensed and converting to FASTA format with ( The adaptor-trimmed sRNA-Seq reads were then aligned to the Arabidopsis thaliana Col-0 genome (TAIR10) including sRNA spike-ins using the Bowtie short-read aligner27 with the options –v 0 –m 100 requiring perfect matches and allowing up to 100 alignments per read (Supplementary Tables 1 and 2). Read numbers per locus were normalized for the number of times they matched the Col-0/spike-in composite genome with ( Reads mapping to miRNA genes, tasiRNA genes, transposons and common 13 nt sequences of the sRNA spike-ins were then grouped together with (, and reads mapping to miRNA and tasiRNA genes were further organized with ( and, respectively). Small RNA-Seq reads were assigned to mature miRNAs or tasiRNAs if they were 20–22 nt long and contained within ±2 nt of the sense strand of the miRNA or tasiRNA according to annotations in miRBase2128 and Allen et al.29, respectively. Values for individual miRNAs or tasiRNAs belonging to common families were then added together to obtain the total amount of reads for individual families. Small RNA reads which were either 20–22 nt or 23–24 nt long and overlapped either strand of TAIR10 annotated transposons (i.e. transposable elements and transposable element genes) were grouped according to the transposon that they mapped to, and are referred to as siRNAs. miRNAs, tasiRNAs, 20–22 nt siRNAs and 23–24 nt siRNAs were grouped together based on the above criteria using ( and, and the levels of miRNAs and tasiRNAs coming from each annotated precursor was quantified with ( Python scripts were used to quantify the RPM levels for 18–30 base reads as well as the base frequencies at each position with (

Paired-end mRNA-Seq reads were aligned to the Arabidopsis thaliana Col-0 genome (TAIR10) and ERCC spike-ins using RSEM30 with default parameters. ERCC spike-in and Araport1131 transcript models were then quantified and recorded in (

Statistical analysis

Statistical analyses were performed and graphics were generated with R32. Biological replicates were included for each sRNA-Seq and mRNA-Seq dataset, and were defined as independently acquired pooled collections of tissues from separate plants. R scripts used to generate all the figures used in this study are publicly available at


  1. 1.

    Meyer, S. U., Pfaffl, M. W. & Ulbrich, S. E. Normalization strategies for microRNA profiling experiments: a ‘normal’ way to a hidden layer of complexity? Biotechnol Lett 32, 1777–1788 (2010).

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Malone, C. D. et al. Specialized piRNA Pathways Act in Germline and Somatic Tissues of the Drosophila Ovary. Cell 137, 522–535 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Farh, K. K. et al. The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science 310, 1817–1821 (2005).

    ADS  CAS  Article  PubMed  Google Scholar 

  4. 4.

    Stark, A., Brennecke, J., Bushati, N., Russell, R. B. & Cohen, S. M. Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3′UTR evolution. Cell 123, 1133–1146 (2005).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Breakfield, N. W. et al. High-resolution experimental and computational profiling of tissue-specific known and novel miRNAs in Arabidopsis. Genome Research 22, 163–176 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Rajagopalan, R., Vaucheret, H., Trejo, J. & Bartel, D. P. A diverse and evolutionarily fluid set of microRNAs in. Genes & Development 20, 3407, doi:10.1101/gad.1476406 (2006).

    CAS  Article  Google Scholar 

  7. 7.

    Martínez, G., Panda, K., Kohler, C. & Slotkin, R. K. Silencing in sperm cells is directed by RNA movement from the surrounding nurse cell. Nature Publishing Group 2, 1603, doi:10.1038/nplants.2016.30 (2016).

  8. 8.

    Fahlgren, N. et al. Computational and analytical framework for small RNA profiling by high-throughput sequencing. RNA 15, 992–1002 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Powers, J. T. et al. Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma. Nature 535, 246–251 (2016).

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Locati, M. D. et al. Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization. Nucleic Acids Research 43, e89–e89 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Hafner, M. et al. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA 17, 1697–1712 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Jayaprakash, A. D., Jabado, O., Brown, B. D. & Sachidanandam, R. Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Research 39, e141–e141 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Sorefan, K. et al. Reducing ligation bias of small RNAs in libraries for next generation sequencing. Silence 3, 1–1 (2012).

    Article  Google Scholar 

  14. 14.

    Yu, B. et al. Methylation as a crucial step in plant microRNA biogenesis. Science 307, 932–935 (2005).

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nature Reviews Genetics 15, 394–408 (2014).

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Gasciolli, V., Mallory, A. C., Bartel, D. P. & Vaucheret, H. Partially Redundant Functions of Arabidopsis DICER-like Enzymes and a Role for DCL4 in Producing trans-Acting siRNAs. Current Biology 15, 1494–1500 (2005).

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Henderson, I. R. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet 38, 721–725 (2006).

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Howell, M. D. et al. Genome-Wide Analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 Pathway in Arabidopsis Reveals Dependency on miRNA- and tasiRNA-Directed Targeting. The Plant Cell 19, 926–942 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Yoshikawa, M. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes & Development 19, 2164–2175 (2005).

    CAS  Article  Google Scholar 

  20. 20.

    Xie, Z. et al. Genetic and Functional Diversification of Small RNA Pathways in Plants. PLoS Biology 2, 0642–0652 (2004).

    CAS  Article  Google Scholar 

  21. 21.

    Yu, B. et al. siRNAs compete with miRNAs for methylation by HEN1 in Arabidopsis. Nucleic Acids Research 38, 5844–5850 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Yoshikawa, M., Peragine, A., Park, M. Y. & Poethig, R. S. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes & Development 19, 2164–2175 (2005).

    CAS  Article  Google Scholar 

  23. 23.

    Addo-Quaye, C., Eshoo, T. W., Bartel, D. P. & Axtell, M. J. Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Current Biology 18, 758–762 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Addo-Quaye, C., Miller, W. & Axtell, M. J. CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics 25, 130–131 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms for Molecular Biology 6, 1–14 (2011).

    Article  Google Scholar 

  26. 26.

    Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Meth 10, 1096–1098 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Kozomara, A. & Griffiths-Jones, S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Research 42, D68–D73 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. microRNA-Directed Phasing during Trans-Acting siRNA Biogenesis in Plants. Cell 121, 207–221 (2005).

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Cheng, C.-Y. et al. Araport11: a complete reannotation of the Arabidopsis thaliana reference genome. The Plant Journal 89, 789–804 (2017).

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    R. Core Team (2016). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. (2016).

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We thank M. Kellner and M. Schon for critical reading of the manuscript. High-throughput sequencing of sRNA-Seq and mRNA-Seq libraries was performed at the VBCF NGS Unit ( This work was supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 637888) and the Austrian Science Fund (FWF) SFB RNA-REG F 43–24 (to M.D.N.).

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M.D.N. conceived the idea, analyzed the data and wrote the manuscript. S.L. generated mRNA-Seq and sRNA-Seq data. B.E. contributed significantly to the development of this study. All authors discussed the results and commented on the manuscript.

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Correspondence to Michael D. Nodine.

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S.L., B.E. and M.D.N. are employed by the Gregor Mendel Institute, a non-profit research organization, that has filed a patent application on the use of the small RNA spike-ins described in this work.

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Lutzmayer, S., Enugutti, B. & Nodine, M.D. Novel small RNA spike-in oligonucleotides enable absolute normalization of small RNA-Seq data. Sci Rep 7, 5913 (2017).

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