Relative fluorescence units
Encyclopedia
The terms "relative fluorescence units" (RFU) and "RFU peak" refer to measurements in electrophoresis
methods, such as for DNA
analysis. A "relative fluorescence unit" is a unit of measurement
used in analysis which employs fluorescence
detection. Fluorescence is detected using a charged coupled device (CCD) array, when the labeled fragments, which are separated within a capillary by using electrophoresis, are energized by laser
light and travel across the detection window. A computer program measures the results, determining the quantity or size of the fragments, at each data point, from the level of fluorescence intensity. Samples which contain higher quantities of amplified DNA will have higher corresponding RFU values.
An "RFU peak" is a relative maximum point along a graph of the analyzed data. The data can be normalized to DNA input or additional normalizing genes. The RFU heights can range from 0 to 2000 or more.
(PCR). Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate
with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotide
s that are labeled with a fluorescent
reporter which permits detection only after hybridization of the probe with its complementary DNA target. Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA
and Non-coding RNA
in cells or tissues.
it can be difficult to separate a true low-level RFU peak from signal noise or other technical artefacts. As a result, many forensic DNA laboratories have set minimum RFU peak-height levels in "scoring" the analysis of allele
s.
There are no firm industry-wide rules for establishing minimum RFU threshold values. Each laboratory, in general, has established its own threshold levels as one aspect of its particular validation procedure. Many laboratories have established both lower and upper thresholds for data interpretation, as a window of minimum and maximum readings.
Some threshold levels can be derived experimentally based on the equipment's known signal-to-noise ratio
s, or a threshold can be defined to match published data or the manufacturer specifications. The company which sells the most widely used equipment for STR typing, Applied Biosystems, Inc. (ABI), has recommended a peak-height minimum of 150 RFU, advising how peaks below that level should be judged with caution. However, many forensic laboratories which have ABI systems have defined lower thresholds, often only 50 to 100 RFU, as determined by their own studies.
Many different factors can affect a laboratory's choice of thresholds. For instance, there might be regulatory guidelines in specific jurisdictions. Also, different kinds of instruments vary in sensitivity (such as slab gel instruments being less sensitive than capillary electrophoresis
(CE) instruments). Individual instruments, of a particular model type, have also been known to differ in performance (e.g. differences among various ABI 310 units, all of the same model). Capillary electrophoresis instruments generally provide better resolution compared gel-based systems, as well having better sensitivity. In addition, some laboratories have set different threshold standards depending on which instruments in the lab are used for an analysis.
Setting an upper maximum threshold is critical when analyzing DNA data within high quantity samples. Samples with large amounts of amplified DNA will report high RFU levels that might oversaturate an instrument's sensitivity to measure the results. In such cases, an accurate measurement of the relative peak heights and/or areas might be unattainable. Oversaturation can be a problem when analyzing mixed samples.
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
methods, such as for DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
analysis. A "relative fluorescence unit" is a unit of measurement
Measurement
Measurement is the process or the result of determining the ratio of a physical quantity, such as a length, time, temperature etc., to a unit of measurement, such as the metre, second or degree Celsius...
used in analysis which employs fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
detection. Fluorescence is detected using a charged coupled device (CCD) array, when the labeled fragments, which are separated within a capillary by using electrophoresis, are energized by laser
Laser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
light and travel across the detection window. A computer program measures the results, determining the quantity or size of the fragments, at each data point, from the level of fluorescence intensity. Samples which contain higher quantities of amplified DNA will have higher corresponding RFU values.
An "RFU peak" is a relative maximum point along a graph of the analyzed data. The data can be normalized to DNA input or additional normalizing genes. The RFU heights can range from 0 to 2000 or more.
DNA PCR analysis
The RFU measurements are used, for DNA profiling, in a real-time polymerase chain reactionReal-time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction , is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule...
(PCR). Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate
Intercalation (chemistry)
In chemistry, intercalation is the reversible inclusion of a molecule between two other molecules . Examples include DNA intercalation and graphite intercalation compounds.- DNA intercalation :...
with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
s that are labeled with a fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
reporter which permits detection only after hybridization of the probe with its complementary DNA target. Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
and Non-coding RNA
Non-coding RNA
A non-coding RNA is a functional RNA molecule that is not translated into a protein. Less-frequently used synonyms are non-protein-coding RNA , non-messenger RNA and functional RNA . The term small RNA is often used for short bacterial ncRNAs...
in cells or tissues.
RFU peak thresholds
The RFU peak height depends on the amount of DNA being analyzed. When the amount of DNA is very low, thenit can be difficult to separate a true low-level RFU peak from signal noise or other technical artefacts. As a result, many forensic DNA laboratories have set minimum RFU peak-height levels in "scoring" the analysis of allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
s.
There are no firm industry-wide rules for establishing minimum RFU threshold values. Each laboratory, in general, has established its own threshold levels as one aspect of its particular validation procedure. Many laboratories have established both lower and upper thresholds for data interpretation, as a window of minimum and maximum readings.
Some threshold levels can be derived experimentally based on the equipment's known signal-to-noise ratio
Signal-to-noise ratio
Signal-to-noise ratio is a measure used in science and engineering that compares the level of a desired signal to the level of background noise. It is defined as the ratio of signal power to the noise power. A ratio higher than 1:1 indicates more signal than noise...
s, or a threshold can be defined to match published data or the manufacturer specifications. The company which sells the most widely used equipment for STR typing, Applied Biosystems, Inc. (ABI), has recommended a peak-height minimum of 150 RFU, advising how peaks below that level should be judged with caution. However, many forensic laboratories which have ABI systems have defined lower thresholds, often only 50 to 100 RFU, as determined by their own studies.
Many different factors can affect a laboratory's choice of thresholds. For instance, there might be regulatory guidelines in specific jurisdictions. Also, different kinds of instruments vary in sensitivity (such as slab gel instruments being less sensitive than capillary electrophoresis
Capillary electrophoresis
Capillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...
(CE) instruments). Individual instruments, of a particular model type, have also been known to differ in performance (e.g. differences among various ABI 310 units, all of the same model). Capillary electrophoresis instruments generally provide better resolution compared gel-based systems, as well having better sensitivity. In addition, some laboratories have set different threshold standards depending on which instruments in the lab are used for an analysis.
Setting an upper maximum threshold is critical when analyzing DNA data within high quantity samples. Samples with large amounts of amplified DNA will report high RFU levels that might oversaturate an instrument's sensitivity to measure the results. In such cases, an accurate measurement of the relative peak heights and/or areas might be unattainable. Oversaturation can be a problem when analyzing mixed samples.