Reference genes in real-time PCR

Bartłomiej Kozera , Marcin Rapacz


This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.
Author Bartłomiej Kozera
Bartłomiej Kozera,,
, Marcin Rapacz (FoAE / DoPP)
Marcin Rapacz,,
- Department of Plant Physiology
Journal seriesJournal of Applied Genetics, ISSN 1234-1983, (A 20 pkt)
Issue year2013
Publication size in sheets0.75
Keywords in EnglishGene expression, Housekeeping genes, Reference gene, Selection, Validation
ASJC Classification1311 Genetics; 2700 General Medicine
Internal identifierWRE/2013/149
Languageen angielski
Score (nominal)20
Score sourcejournalList
Publication indicators WoS Citations = 286; Scopus SNIP (Source Normalised Impact per Paper): 2013 = 0.96; WoS Impact Factor: 2013 = 1.902 (2) - 2013=1.74 (5)
Citation count*78 (2016-03-21)
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* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.
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