science

RNA spike-in

RNA spike-in is a science topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand RNA spike-in rather than just read about it. In short: 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. A spike-in is designed to bind to a DNA molecule with a matching sequence, known as a control probe.

Key takeaways

RNA spike-in belongs to science; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect RNA spike-in to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of RNA spike-in from memory before moving on to harder problems.

Reference excerpt

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. A spike-in is designed to bind to a DNA 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 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 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. 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 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). RNA can be produced in vitro (cell free) using RNA polymerase and DNA with the desired sequence. Large scale biotech manufacturers produce RNA synthetically via high-throughput techniques and provide solutions of RNA spike-ins at predetermined concentration. Bacteria containing DNA (usually on plasmids) 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 surfaces, usually a small chip, to which short DNA polymers 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. Bound transcripts can be detected, indicating the presence of RNA with the corresponding sequence. 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. 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.

Sequencing

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

Illustrations

RNA spike-in: 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. — 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.

Worked examples

Example 1 — a first encounter with RNA spike-in

Start with the simplest possible case. Write down what RNA spike-in claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In science, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to RNA spike-in before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about RNA spike-in ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of RNA spike-in

In research: RNA spike-in appears in science research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses RNA spike-in in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: RNA spike-in is common in secondary-school and first-year university syllabi. It links to neighbouring topics Microarrays, RNA, so understanding it makes those chapters shorter.
In everyday life: Look for RNA spike-in outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

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How to study RNA spike-in in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what RNA spike-in means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
Explain RNA spike-in out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is RNA spike-in in simple terms?

Why does RNA spike-in matter?

Because it connects several science ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study RNA spike-in?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on RNA spike-in.