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]
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]
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]