2
Genome
Biology
Vol 3 No 7
Vandesompele
et al.
competitive RT-PCR Nevertheless, these new approachesrequire the same kind of normalization as the traditionalmethods of mRNA quantificationSeveral variables need to be controlled for in gene-expres-sion analysis, such as the amount of starting material, enzy-matic efficiencies, and differences between tissues or cells inoverall transcriptional activity Various strategies have beenapplied to normalize these variations Under controlled con-ditions of reproducible extraction of good-quality RNA, thegene transcript number is ideally standardized to thenumber of cells, but accurate enumeration of cells is oftenprecluded, for example when starting with solid tissue Another frequently applied normalization scalar is the RNA mass quantity, especially in northern blot analysis There areseveral arguments against the use of mass quantity Thequality of RNA and related efficiency of the enzymatic reac-tions are not taken into account Moreover, in someinstances it is impossible to quantify this parameter, forexample, when only minimal amounts of RNA are availablefrom microdissected tissues Probably the strongest argu-ment against the use of total RNA mass for normalization isthe fact that it consists predominantly of rRNA molecules,and is not always representative of the mRNA fraction This was recently evidenced by a significant imbalance betweenrRNA and mRNA content in approximately 75% of mammary adenocarcinomas [5] Also, it has been reportedthat rRNA transcription is affected by biological factors anddrugs [6-8] .urther drawbacks to the use of 18S or 28SrRNA molecules as standards are their absence in purifiedmRNA samples, and their high abundance compared totarget mRNA transcripts The latter makes it difficult toaccurately subtract the baseline value in real-time RT-PCR data analysisTo date, internal control genes are most frequently used tonormalize the mRNA fraction This internal control - oftenreferred to as a housekeeping gene - should not vary in thetissues or cells under investigation, or in response to experi-mental treatment However, many studies make use of theseconstitutively expressed control genes without proper vali-dation of their presumed stability of expression But the lit-erature shows that housekeeping gene expression - althoughoccasionally constant in a given cell type or experimentalcondition - can vary considerably (reviewed in [9-12]) Withthe increased sensitivity, reproducibility and large dynamicrange of real-time RT-PCR methods, the requirements for aproper internal control gene have become increasingly strin-gent In this study, we carried out an extensive evaluation of 10 commonly used housekeeping genes in 13 differenthuman tissues, and outlined a procedure for calculating anormalization factor based on multiple control genes formore accurate and reliable normalization of gene-expressiondata .urthermore, this normalization factor was validatedin a comparative study with frequently applied microarray scaling factors using publicly available microarray data
Results
Expression profiling of housekeeping genes
Primers were designed for ten commonly used housekeepinggenes (
ACTB
,
B2M
,
GAPD
,
HMBS
,
HPRT1
,
RPL13A
,
SDHA
,
TBP
,
UBC
and
YWHAZ
) (see Table1 for full gene name,accession number, function, chromosomal localization, alias,existence of processed pseudogenes, and indication thatprimers span an intron; see Table2 for primer sequences)Special attention was paid to selecting genes that belong todifferent functional classes, which significantly reduces thechance that genes might be co-regulated The expressionlevel of these 10 internal control genes was determined in 34neuroblastoma cell lines (independently prepared in differ-ent labs from different patients), 20 short-term culturednormal fibroblast samples from different individuals, 13normal leukocyte samples, 9 normal bone-marrow samples,and 9 additional normal human tissues from pooled organs(heart, brain, fetal brain, lung, trachea, kidney, mammary gland, small intestine and uterus) The raw expression values are available as a tab-delimited file (see Additionaldata files)
Single control normalization error
To determine the possible errors related to the commonpractice of using only one housekeeping gene for normaliza-tion, we calculated the ratio of the ratios of two control genesin two different samples (from the same tissue panel) andtermed it the single control normalization error,
E
(seeMaterials and methods) .or two ideal internal control genes(constant ratio between the genes in all samples),
E
equals 1In practice, observed
E
values are larger than 1 and consti-tute the erroneous
E
-fold expression difference between twosamples, depending on the particular housekeeping geneused for normalization
E
values were calculated for all 45two-by-two combinations of control genes and 865 two-by-two sample combinations within the available tissue panels(neuroblastoma, fibroblast, leukocyte, bone marrow and aseries of normal tissues from Clontech; that is, a total of 38,925 data points) (.igure1) In addition, the systematicerror distribution was calculated by analysis of repeatedruns of the same control gene The average 75th and 90thpercentile
E
values are 30 (range 21-39), and 64 (range30-109), respectively
Gene-stability measure and ranking of selectedhousekeeping genes
It is generally accepted that gene-expression levels should benormalized by a carefully selected stable internal controlgene However, to validate the presumed stable expression of a given control gene, prior knowledge of a reliable measure tonormalize this gene in order to remove any nonspecific varia-tion is required To address this circular problem, we devel-oped a gene-stability measure to determine the expressionstability of control genes on the basis of non-normalizedexpression levels This measure relies on the principle thatthe expression ratio of two ideal internal control genes is
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