# Relative fluorescence units

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The terms ***relative fluorescence units*** (RFU) and ***RFU peak*** refer to measurements in [electrophoresis](/source/Electrophoresis) methods, such as for [DNA](/source/DNA) analysis. A *relative fluorescence unit* is a unit of [measurement](/source/Measurement) used in analysis which employs [fluorescence](/source/Fluorescence) detection.[1] Fluorescence is detected using a [charge-coupled device](/source/Charge-coupled_device) (CCD) array, when the labeled fragments, which are separated within a capillary by using electrophoresis, are energized by [laser](/source/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.[1] Samples which contain higher quantities of amplified DNA will have higher corresponding RFU values.[2][3]

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 several thousands.

## DNA PCR analysis

The RFU measurements are used, for [DNA profiling](/source/DNA_profiling), in a [real-time polymerase chain reaction](/source/Real-time_polymerase_chain_reaction) (PCR). Two common methods for detection of products in real-time PCR are: (1) non-specific [fluorescent dyes](/source/Fluorescent_dye) that [intercalate](/source/Intercalation_(biochemistry)) with any double-stranded DNA, and (2) sequence-specific [DNA probes](/source/DNA_probe) consisting of [oligonucleotides](/source/Oligonucleotide) that are labeled with a [fluorescent](/source/Fluorescence) reporter which permits detection only after [hybridization](/source/Nucleic_acid_hybridisation) of the probe with its complementary DNA target. Frequently, real-time PCR is combined with [reverse transcription](/source/Reverse_transcription) to quantify [messenger RNA](/source/Messenger_RNA) and [Non-coding RNA](/source/Non-coding_RNA) 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, then it can be difficult to separate a true low-level RFU peak from [signal noise](/source/Signal_noise) or other technical [artifacts](/source/Artifact_(error)).[4] As a result, many forensic DNA laboratories have set minimum RFU peak-height levels in *scoring* the analysis of [alleles](/source/Allele).[4]

There are no firm industry-wide rules for establishing minimum RFU threshold values.[4] 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.[4]

Some threshold levels can be derived experimentally based on the equipment's known [signal-to-noise ratios](/source/Signal-to-noise_ratio), or a threshold can be defined to match published data or the manufacturer specifications.[4] 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.[4]

Many different factors can affect a laboratory's choice of thresholds.[4] For instance, there might be regulatory guidelines in specific jurisdictions. Also, different kinds of instruments vary in sensitivity (such as [slab gel](/source/Slab_gel) instruments being less sensitive than [capillary electrophoresis](/source/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.[4]

Setting an upper maximum threshold is critical when analyzing DNA data within high quantity samples.[4] 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.[4]

## References

1. "Relative fluorescence unit (RFU)", DNA.gov: Glossary, April 2011, webpage: [DNA-gov-RFU](http://www.dna.gov/glossary/).

1. "Thresholds", NFSTC.org, 2010, webpage: [\[1\]](http://www.nfstc.org/pdi/Subject06/pdi_s06_m02_02.htm).

1. Gertsch J, Güttinger M, Sticher O, Heilmann J (2002). "Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt-PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds". *Pharm Res*. **19** (8): 1236–1243. [doi:10.1023/A:1019818814336](https://doi.org/10.1023/A:1019818814336). [PMID 12240952](https://pubmed.ncbi.nlm.nih.gov/12240952). [S2CID 17465967](https://api.semanticscholar.org/CorpusID:17465967)

1. "Advancing Justice Through DNA Technology", [National Forensic Science Technology Center](/source/National_Forensic_Science_Technology_Center) (NFSTC), 2011, web: [NFS2](http://www.nfstc.org/pdi/Subject06/pdi_s06_m02_02.htm).

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