Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations
Abstract
The stability of standard gene expression is an elementary prerequisite for internal standardisation of target gene expression data and many so called housekeeping genes with assumed stable expression can exhibit either up- or down-regulation under some experimental conditions. The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points. The BestKeeper software tool was validated on four housekeeping genes and 10 members of the somatotropic axis differentially expressed in bovine corpora lutea total RNA. The BestKeeper application and necessary information about data processing and handling can be downloaded on http://www.wzw.tum.de/gene-quantification/bestkeeper.html.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
BestKeeper software was used to search for stable reference genes among all genes tested.
“The BestKeeper software [52] was used to search for stable reference genes among all genes tested.”
From Relative expression of genes of terpene metabolism in different tissues of Artemisia annuaL (2011) · cites this paper as [52]
Cites
A new mathematical model for relative quantification in real-time RT-PCR
Pfaffl, M. W. (2001) · Nucleic Acids Research · doi:10.1093/nar/29.9.e45 · Cited by 34,610 · see citing works
Self-citationVandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., & Speleman, F. (2002) · Genome Biology · doi:10.1186/gb-2002-3-7-research0034 · Cited by 20,038 · see citing works
Pfaffl, M. W. (2002) · Nucleic Acids Research · doi:10.1093/nar/30.9.e36 · Cited by 7,377 · see citing works
Self-citationChomczyński, P. (1993) · PubMed · Cited by 2,788 · see citing works
Housekeeping genes as internal standards: use and limits
Thellin, O., Zorzi, W., Lakaye, B., Borman, B. D., Coumans, B., Hennen, G. P., Grisar, T. M., Igout, A., & Heinen, E. (1999) · Journal of Biotechnology · doi:10.1016/s0168-1656(99)00163-7 · Cited by 1,534 · see citing works
Processing of gene expression data generated by quantitative real-time RT-PCR.
Muller, P. Y., Janovjak, H., Miserez, A. R., & Dobbie, Z. (2002) · PubMed · Cited by 1,410 · see citing works
Schmittgen, T. D., & Zakrajsek, B. A. (2000) · Journal of Biochemical and Biophysical Methods · doi:10.1016/s0165-022x(00)00129-9 · Cited by 1,252 · see citing works
The LightCycler TM : A Microvolume Multisample Fluorimeter with Rapid Temperature Control
Wittwer, C. T., Ririe, K. M., Andrew, R., David, D., Gundry, R. A., & Balis, U. J. (1997) · BioTechniques · doi:10.2144/97221pf02 · Cited by 901 · see citing works
Quantification on the LightCycler
Rasmussen, R. P. (2001) · Springer eBooks · doi:10.1007/978-3-642-59524-0_3 · Cited by 894 · see citing works
Control Selection for RNA Quantitation
Suzuki, T., Higgins, P. J., & Crawford, D. R. (2000) · BioTechniques · doi:10.2144/00292rv02 · Cited by 878 · see citing works
Rapid Cycle Real-Time PCR: Methods and Applications
Wittwer, C. T. (2001) · Springer eBooks · doi:10.1007/978-3-642-59524-0_1 · Cited by 636 · see citing works
Liu, W., & Saint, D. A. (2002) · Analytical Biochemistry · doi:10.1006/abio.2001.5530 · Cited by 521 · see citing works
Standardized determination of real-time PCR efficiency from a single reaction set-up
Tichopád, A. (2003) · Nucleic Acids Research · doi:10.1093/nar/gng122 · Cited by 456 · see citing works
Normalization strategies for cDNA microarrays
Schuchhardt, J. (2000) · Nucleic Acids Research · doi:10.1093/nar/28.10.e47 · Cited by 332 · see citing works
Pfaffl, M. W., & Hageleit, M. (2001) · Biotechnology Letters · doi:10.1023/a:1005658330108 · Cited by 300 · see citing works
Self-citationPfaffl, M. W., Georgieva, T. M., Georgiev, I. P., Ontsouka, E. C., Hageleit, M., & Blum, J. W. (2002) · Domestic Animal Endocrinology · doi:10.1016/s0739-7240(01)00128-x · Cited by 172 · see citing works
Self-citationFoss, D. L., Baarsch, M. J., & Murtaugh, M. P. (1998) · Animal Biotechnology · doi:10.1080/10495399809525893 · Cited by 147 · see citing works
Serazin-Leroy, V., Denis-Henriot, D., Morot, M., de Mazancourt, P., & Giudicelli, Y. (1998) · Molecular and Cellular Probes · doi:10.1006/mcpr.1998.0182 · Cited by 55 · see citing works
FROM THE DECLINE AND FALL OF PROTEIN CHEMISTRY TO PROTEOMICS
Chakravarti, D. N. (2002) · BioTechniques · doi:10.2144/mar0201 · Cited by 31 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., & Vandesompele, J. (2007) · Genome Biology · doi:10.1186/gb-2007-8-2-r19 · Cited by 4,023 · see citing works
Quantification of mRNA using real-time RT-PCR
Nolan, T., Hands, R. E., & Bustin, S. A. (2006) · Nature Protocols · doi:10.1038/nprot.2006.236 · Cited by 2,247 · see citing works
Twenty-Five Years of Quantitative PCR for Gene Expression Analysis
VanGuilder, H. D., Vrana, K. E., & Freeman, W. M. (2008) · BioTechniques · doi:10.2144/000112776 · Cited by 1,246 · see citing works
Gutierrez, L., Mauriat, M., Guénin, S., Pelloux, J., Lefebvre, J., Louvet, R., Rusterucci, C., Moritz, T., Guerineau, F., Bellini, C., et al. (2008) · Plant Biotechnology Journal · doi:10.1111/j.1467-7652.2008.00346.x · Cited by 696 · see citing works
Kuang, J., Yan, X., Genders, A. J., Granata, C., & Bishop, D. J. (2018) · PLOS ONE · doi:10.1371/journal.pone.0196438 · Cited by 207 · see citing works
Park, S.-C., Kim, Y.-H., Ji, C. Y., Park, S., Jeong, J. C., Lee, H.-S., & Kwak, S.-S. (2012) · PLoS ONE · doi:10.1371/journal.pone.0051502 · Cited by 160 · see citing works
Assessing the role of muscle protein breakdown in response to nutrition and exercise in humans.
Tipton, K. D., Hamilton, D. L., & Gallagher, I. J. (2018) · Sports Medicine · doi:10.1007/s40279-017-0845-5 · Cited by 152 · see citing works
Relative expression of genes of terpene metabolism in different tissues of Artemisia annuaL
Olofsson, L., Engström, A., Lundgren, A., & Brodelius, P. E. (2011) · BMC Plant Biology · doi:10.1186/1471-2229-11-45 · Cited by 144 · see citing works
Afatinib restrains K-RAS–driven lung tumorigenesis
Moll, H. P., Pranz, K., Musteanu, M., Grabner, B., Hruschka, N., Mohrherr, J., Aigner, P., Stiedl, P., Brcic, L., Laszlo, V., et al. (2018) · Science Translational Medicine · doi:10.1126/scitranslmed.aao2301 · Cited by 125 · see citing works
Dergunova, L. V., Filippenkov, I. B., Stavchansky, V. V., Denisova, A. E., Yuzhakov, V. V., Mozerov, S. A., Gubsky, L. V., & Limborska, S. A. (2018) · BMC Genomics · doi:10.1186/s12864-018-5039-5 · Cited by 70 · see citing works
Aberrant Wnt signaling pathway in medial temporal lobe structures of Alzheimer’s disease
Riise, J., Plath, N., Pakkenberg, B., & Parachikova, A. (2015) · Journal of Neural Transmission · doi:10.1007/s00702-015-1375-7 · Cited by 66 · see citing works
Verma, P., Sharma, A., Sodhi, M., Thakur, K., Kataria, R. S., Niranjan, S. K., Bharti, V. K., Kumar, P., Giri, A., Kalia, S., et al. (2018) · Scientific Reports · doi:10.1038/s41598-018-25736-7 · Cited by 63 · see citing works
Chaturvedi, V., Naskar, D., Kinnear, B. F., Grenik, E., Dye, D. E., Grounds, M. D., Kundu, S. C., & Coombe, D. R. (2017) · Journal of Tissue Engineering and Regenerative Medicine · doi:10.1002/term.2227 · Cited by 50 · see citing works
Bile acid mediated effects on gut integrity and performance of early weaned piglets
de Diego-Cabero, N., Mereu, A., Menoyo, D., Holst, J. J., & Ipharraguerre, I. R. (2015) · BMC Veterinary Research · doi:10.1186/s12917-015-0425-6 · Cited by 35 · see citing works
Comparative analysis of algorithms for integration of copy number and expression data
Louhimo, R., Lepikhova, T., Monni, O., & Hautaniemi, S. (2012) · Nature Methods · doi:10.1038/nmeth.1893 · Cited by 30 · see citing works
Filippenkov, I. B., Stavchansky, V. V., Denisova, A. E., Yuzhakov, V. V., Sevan’kaeva, L. E., Sudarkina, O. Y., Dmitrieva, V. G., Gubsky, L. V., Myasoedov, N. F., Limborska, S. A., et al. (2020) · Genes · doi:10.3390/genes11060681 · Cited by 29 · see citing works
“Calculations were performed using BestKeeper, version 1 (gene-quantification, Freising-Weihenstephan, Bavaria, Germany) [41] and Relative Expression Software Tool (REST) 2005 software (gene-quantification, Freising-Weihenstephan, Bavaria, Germany) [42].”
MethodA 3.2 Mb deletion on 18q12 in a patient with childhood autism and high-grade myopia
Gilling, M., Lauritsen, M. B., Møller, M., Henriksen, K. F., Vicente, A., Oliveira, G., Cintin, C., Eiberg, H., Andersen, P. S., Mors, O., et al. (2008) · European Journal of Human Genetics · doi:10.1038/sj.ejhg.5201985 · Cited by 18 · see citing works
Sudarkina, O. Y., Filippenkov, I. B., Stavchansky, V. V., Denisova, A. E., Yuzhakov, V. V., Sevan’kaeva, L. E., Valieva, L. V., Remizova, J. A., Dmitrieva, V. G., Gubsky, L. V., et al. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms22126179 · Cited by 15 · see citing works
“The PCR efficiencies were assessed using the amplification of a series of standard dilutions of cDNAs and computed using REST software [54].”
MethodAtlantic salmon (Salmo salar L.) as a marine functional source of gamma-tocopherol
Menoyo, D., Sanz-Bayón, C., Nessa, A., Esatbeyoglu, T., Faizan, M., Pallauf, K., De Diego, N., Wagner, A., Ipharraguerre, I., Stubhaug, I., et al. (2014) · Marine Drugs · doi:10.3390/md12125944 · Cited by 12 · see citing works
“The geometric mean of the reference genes β-actin and EF-1α was used to correct Ct values of target genes [47].”
Rangel-Zúñiga, O. A., Corina, A., Lucena-Porras, B., Cruz-Teno, C., Gómez-Delgado, F., Jiménez-Lucena, R., Alcalá-Díaz, J. F., Haro-Mariscal, C., Yubero-Serrano, E. M., Delgado-Lista, J., et al. (2016) · Experimental Gerontology · doi:10.1016/j.exger.2016.07.015 · Cited by 11 · see citing works
Shimada, H., Obayashi, T., Takahashi, N., Matsui, M., & Sakamoto, A. (2010) · Plant Methods · doi:10.1186/1746-4811-6-29 · Cited by 10 · see citing works
Díaz‐Lara, J., Reisman, E., Botella, J., Probert, B., Burke, L. M., Bishop, D. J., & Lee, M. J. (2024) · Acta Physiologica · doi:10.1111/apha.14215 · Cited by 10 · see citing works
Duenser, A., Singh, P., Lecaudey, L. A., Sturmbauer, C., Albertson, C., Gessl, W., & Ahi, E. P. (2022) · bioRxiv · doi:10.1101/2022.01.13.476207 · Cited by 1 · see citing works
- Authors
- Pfaffl, M. W., Tichopad, A., Prgomet, C., & Neuvians, T. P.
- Affiliations
- Technical University of Munich (DE)
- ORCID
- Pfaffl, Michael W.
- Venue
- Biotechnology Letters
- Published
- 2004-03
- Volume
- 26
- Issue
- 6
- Pages
- 509-515
- ISSN
- 0141-5492 (ISSN-L)
- 1573-6776
- OpenAlex
- W2099824652
- Cited by
- 5,065
- References
- 19
- Citations by year
- 2026: 180 · 2025: 273 · 2024: 289 · 2023: 287 · 2022: 311 · 2021: 369 · 2020: 391 · 2019: 379 · 2018: 372 · 2017: 333 · 2016: 321 · 2015: 326 · 2014: 282 · 2013: 223 · 2012: 188
- FWCI
- 14.92
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Biotechnology Letters (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Keywords
- housekeeping genes, gene expression stability, two-particle correlations, differentially expressed genes, sample integrity, crossing points, somatotropic axis, bovine corpus luteum, gene expression analysis
- MeSH terms
- Animals, Cattle, Corpus Luteum, Female, Genes, RNA, Software, Gene Expression, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review