An RNA spike-in is an

RNA transcript Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules calle ...

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 calle ...

and

quantity Quantity or amount is a property that can exist as a Counting, multitude or Magnitude (mathematics), magnitude, which illustrate discontinuity (mathematics), discontinuity and continuum (theory), continuity. Quantities can be compared in terms o ...

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 a ...

measurements in

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

hybridization assay A hybridization assay comprises any form of quantifiable hybridization ''i.e.'' the quantitative annealing of two complementary strands of nucleic acids, known as nucleic acid hybridization. Overview In the context of biochemistry and drug develop ...

s, such as

DNA microarray A DNA microarray (also commonly known as 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 to ...

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 Causality, cause-and-effect by demonstrating what outcome oc ...

RT-qPCR A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...

, and

RNA-Seq RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...

. A spike-in is designed to bind to a DNA molecule with a matching sequence, known as a

control Control may refer to: Basic meanings Economics and business * Control (management), an element of management * Control, an element of management accounting * Comptroller (or controller), a senior financial officer in an organization * Controllin ...

probe. This process of specific binding is called

hybridization Hybridization (or hybridisation) may refer to: *Hybridization (biology), the process of combining different varieties of organisms to create a hybrid *Orbital hybridization, in chemistry, the mixing of atomic orbitals into new hybrid orbitals *Nu ...

. 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 biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...

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, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids c ...

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 A complement is something that completes something else. Complement may refer specifically to: The arts * Complement (music), an interval that, when added to another, spans an octave ** Aggregate complementation, the separation of pitch-class ...

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 by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...

assay microarrays or

RNA sequencing RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...

(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 Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...

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, and ...

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

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

'' (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 synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the ...

and DNA with the desired sequence. Large scale

biotech Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used b ...

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; how ...

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 one of the State of matter#Four fundamental states, four fundamental states of matter (the others being liquid, gas, and Plasma (physics), plasma). The molecules in a solid are closely packed together and contain the least amount o ...

surfaces, usually a small chip, to which short DNA

polymer A polymer (; Greek '' poly-'', "many" + ''-mer'', "part") is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...

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 synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...

. 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 In signal processing, a signal is a function that conveys information about a phenomenon. Any quantity that can vary over space or time can be used as a signal to share messages between observers. The ''IEEE Transactions on Signal Processing'' ...

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