A spike-in is designed to bind to a DNA molecule with a matching sequence, known as a controlprobe.[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]
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.
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]
Applicationsedit
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 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]
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]
^ abcdefghYang IV (2006). "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. ISBN9780121828165. PMID 16939785.
^ abcdFardin 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. PMC1797020. PMID 17229315.
^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.
^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. PMC2394775. PMID 17594493.
^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. ISBN9780896038226. PMID 11357674.
^ abcdeGillespie, 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.
^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. ISBN9780121828165. PMID 16939785.
^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. Bibcode:1995Sci...270..467S. doi:10.1126/science.270.5235.467. ISSN 0036-8075. PMID 7569999.
spike, transcript, known, sequence, quantity, used, calibrate, measurements, hybridization, assays, such, microarray, experiments, qpcr, three, dimensional, structure, molecule, short, synthetic, polymers, spike, designed, bind, molecule, with, matching, seque. 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 Three dimensional structure of an RNA molecule RNA spike ins are short synthetic RNA polymers 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 Contents 1 History 2 Manufacturing 3 Applications 3 1 DNA microarrays 3 2 Sequencing 4 See also 5 ReferencesHistory editNucleic 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 editMain article Oligonucleotide synthesis 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 edit nbsp 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 edit Main article DNA microarray 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 edit Main article RNA Seq 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 editDNA spiking qPCRReferences edit a b c d e f g h Yang IV 2006 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 a b c d 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 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 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 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 a b c d e 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 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 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 Bibcode 1995Sci 270 467S doi 10 1126 science 270 5235 467 ISSN 0036 8075 PMID 7569999 a b c 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 Retrieved from https en wikipedia org w index php title RNA spike in amp oldid 1209550906, wikipedia, wiki, book, books, library,
