Three-dimensional structure of an RNA molecule. RNA spike-ins are short synthetic RNA polymers.

An RNA spike-in is an RNA transcript of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, RT-qPCR, and RNA-Seq.[1]

A spike-in is designed to bind to a DNA molecule with a matching sequence, known as a control probe.[2][3][4] This process of specific binding is called hybridization. A known quantity of RNA spike-in is mixed with the experiment sample during preparation.[2] The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements of the sample RNA.[2]

History

Nucleic acid hybridization assays have been used for decades to detect specific sequences of DNA or RNA,[5] with a DNA microarray precursor used as early as 1965.[6] In such assays, positive control oligonucleotides are necessary to provide a standard for comparison of target sequence concentration, and to check and correct for nonspecific binding; that is, incidental binding of the RNA to non-complementary DNA sequences.[7] These controls became known as "spike-ins".[1] With the advent of DNA microarray chips in the 1990s[8] and the commercialization of high-throughput methods for sequencing and RNA detection assays, manufacturers of hybridization assay "kits" started to provide pre-developed spike-ins.[1] In the case of gene expression assay microarrays or RNA sequencing (RNA-seq), RNA spike-ins are used.

Manufacturing

RNA spike-ins can be synthesized by any means of creating RNA synthetically, or by using cells to transcribe DNA to RNA in vivo (in cells).[1] RNA can be produced in vitro (cell free) using RNA polymerase and DNA with the desired sequence.[1] Large scale biotech manufacturers produce RNA synthetically via high-throughput techniques and provide solutions of RNA spike-ins at predetermined concentration.[1] Bacteria containing DNA (usually on plasmids) for transcription to spike-ins are also commercially available.[1] The purified RNA can be stored long-term in a buffered solution at low temperature.[1]

Applications

Example of DNA microarray data. The bright spots show locations where hybridization has occurred, indicating that RNA of the corresponding sequence was present in the sample.

DNA microarrays

DNA microarrays are solid surfaces, usually a small chip, to which short DNA polymers of known sequence are covalently bound.[6] When a sample of unknown RNA is flowed over the array, the RNA base pairs with and binds to complementary DNA.[6] Bound transcripts can be detected, indicating the presence of RNA with the corresponding sequence.[6] DNA microarray assays are useful in studies of gene expression, because many of the mRNA transcripts present in a cell can be detected at the same time.[6] RNA spike-ins of known quantity can provide a baseline signal for comparison with the signal from transcripts of unknown quantity, such that the data can be normalized within an array and between different arrays.[2]

Sequencing

RNA sequencing (RNA-Seq) is performed by reverse transcribing RNA to complementary DNA (cDNA) and high-throughput sequencing the cDNA.[9] Such high-throughput methods can be error prone, and known controls are necessary to detect and correct for levels of error.[9] RNA spike-in controls can provide a measure of sensitivity and specificity of an RNA-Seq experiment.[9]

See also

References

  1. 1 2 3 4 5 6 7 8 Yang IV (2006). "[4] Use of External Controls in Microarray Experiments". DNA Microarrays, Part B: Databases and Statistics. Methods in Enzymology. Vol. 411. pp. 50–63. doi:10.1016/S0076-6879(06)11004-6. ISBN 9780121828165. PMID 16939785. {{cite book}}: |journal= ignored (help)
  2. 1 2 3 4 Fardin P, Moretti S, Biasotti B, Ricciardi A, Bonassi S, Varesio L (2007). "Normalization of low-density microarray using external spike-in controls: analysis of macrophage cell lines expression profile". BMC Genomics. 8: 17. doi:10.1186/1471-2164-8-17. PMC 1797020. PMID 17229315.
  3. ↑ Wilkes T, Laux H, Foy CA (2007). "Microarray data quality - review of current developments". OMICS. 11 (1): 1–13. doi:10.1089/omi.2006.0001. PMID 17411392.
  4. ↑ Schuster EF, Blanc E, Partridge L, Thornton JM (2007). "Estimation and correction of non-specific binding in a large-scale spike-in experiment". Genome Biol. 8 (6): R126. doi:10.1186/gb-2007-8-6-r126. PMC 2394775. PMID 17594493.
  5. ↑ Southern, Edwin M. (2001). "DNA Microarrays: History and Overview". DNA Arrays. Methods in Molecular Biology. Vol. 170. Humana Press. pp. 1–15. doi:10.1385/1-59259-234-1:1. ISBN 9780896038226. PMID 11357674.
  6. 1 2 3 4 5 Gillespie, D.; Spiegelman, S. (July 1965). "A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane". Journal of Molecular Biology. 12 (3): 829–842. doi:10.1016/s0022-2836(65)80331-x. ISSN 0022-2836. PMID 4955314.
  7. ↑ Yang, Ivana V. (2006-01-01). "[4] Use of External Controls in Microarray Experiments". DNA Microarrays, Part B: Databases and Statistics. Methods in Enzymology. Vol. 411. Academic Press. pp. 50–63. doi:10.1016/S0076-6879(06)11004-6. ISBN 9780121828165. PMID 16939785.
  8. ↑ Schena, Mark; Shalon, Dari; Davis, Ronald W.; Brown, Patrick O. (1995-10-20). "Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray". Science. 270 (5235): 467–470. doi:10.1126/science.270.5235.467. ISSN 0036-8075. PMID 7569999.
  9. 1 2 3 Jiang, Lichun; Schlesinger, Felix; Davis, Carrie A.; Zhang, Yu; Li, Renhua; Salit, Marc; Gingeras, Thomas R.; Oliver, Brian (2011-09-01). "Synthetic spike-in standards for RNA-seq experiments". Genome Research. 21 (9): 1543–1551. doi:10.1101/gr.121095.111. ISSN 1088-9051. PMC 3166838. PMID 21816910.
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