An RNA spike-in is an

RNA transcript Transcription is the process of copying a segment of DNA into RNA for the purpose of gene expression. Some segments of DNA are transcribed into RNA molecules that can encode proteins, called messenger RNA (mRNA). Other segments of DNA are transc ...

of known

sequence In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is cal ...

and

quantity Quantity or amount is a property that can exist as a multitude or magnitude, which illustrate discontinuity and continuity. Quantities can be compared in terms of "more", "less", or "equal", or by assigning a numerical value multiple of a u ...

used to

calibrate In measurement technology and metrology, calibration is the comparison of measurement values delivered by a device under test with those of a calibration standard of known accuracy. Such a standard could be another measurement device of known ...

measurements in

RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...

hybridization assays, such as

DNA microarray A DNA microarray (also commonly known as a DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or t ...

experiment An experiment is a procedure carried out to support or refute a hypothesis, or determine the efficacy or likelihood of something previously untried. Experiments provide insight into cause-and-effect by demonstrating what outcome occurs whe ...

s, RT-qPCR, and

RNA-Seq RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also k ...

. A spike-in is designed to bind to a

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

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

History

Nucleic acid Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a pentose, 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nuclei ...

hybridization assays have been used for decades to detect specific sequences of DNA or RNA, with a DNA microarray precursor used as early as 1965. In such assays, positive control

oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, Recombinant DNA, research, and Forensic DNA, forensics. Commonly made in the laboratory by Oligonucleotide synthesis, solid-phase ...

s 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 Complement may refer to: The arts * Complement (music), an interval that, when added to another, spans an octave ** Aggregate complementation, the separation of pitch-class collections into complementary sets * Complementary color, in the visu ...

DNA sequences. These controls became known as "spike-ins". With the advent of DNA microarray chips in the 1990s 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. In the case of

gene expression Gene expression is the process (including its Regulation of gene expression, regulation) by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, ...

assay microarrays or

RNA sequencing RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also kn ...

(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 Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, an ...

'' (in cells). RNA can be produced ''

in vitro ''In vitro'' (meaning ''in glass'', or ''in the glass'') Research, studies are performed with Cell (biology), cells or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in ...

'' (cell free) using

RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template. Using the e ...

and DNA with the desired sequence. Large scale

biotech Biotechnology is a multidisciplinary field that involves the integration of natural sciences and engineering sciences in order to achieve the application of organisms and parts thereof for products and services. Specialists in the field are kn ...

manufacturers produce RNA synthetically via high-throughput techniques and provide solutions of RNA spike-ins at predetermined concentration. Bacteria containing DNA (usually on

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...

s) for transcription to spike-ins are also commercially available. The purified RNA can be stored long-term in a buffered solution at low temperature.

Applications

DNA microarrays

DNA microarrays are

solid Solid is a state of matter where molecules are closely packed and can not slide past each other. Solids resist compression, expansion, or external forces that would alter its shape, with the degree to which they are resisted dependent upon the ...

surfaces, usually a small chip, to which short DNA

polymer A polymer () is a chemical substance, substance or material that consists of very large molecules, or macromolecules, that are constituted by many repeat unit, repeating subunits derived from one or more species of monomers. Due to their br ...

s of known sequence are covalently bound. When a sample of unknown RNA is flowed over the array, the RNA base pairs with and binds to

complementary DNA In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...

. Bound transcripts can be detected, indicating the presence of RNA with the corresponding sequence. DNA microarray assays are useful in studies of

, because many of the mRNA transcripts present in a cell can be detected at the same time. RNA spike-ins of known quantity can provide a baseline

signal A signal is both the process and the result of transmission of data over some media accomplished by embedding some variation. Signals are important in multiple subject fields including signal processing, information theory and biology. In ...

for comparison with the signal from transcripts of unknown quantity, such that the data can be normalized within an array and between different arrays.

Sequencing

RNA sequencing (RNA-Seq) is performed by reverse transcribing RNA to

(cDNA) and high-throughput sequencing the cDNA. Such high-throughput methods can be error prone, and known controls are necessary to detect and correct for levels of error. RNA spike-in controls can provide a measure of sensitivity and specificity of an RNA-Seq experiment.

References