1. Normalization strategies for mRNA expression data in cartilage research 
K. Fundel, Ph.D., J. Haag, Ph.D., P.M. Gebhard, M.Sc., R. Zimmer, Ph.D., T. Aigner, M.D., D.Sc. 
Osteoarthritis and Cartilage 
Volume 16, Issue 8, Pages (August 2008)
DOI: /j.joca
Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Effects of normalization. (A) Group-level plots for peripheral (samples 1–21) and central (samples 22–40) late stage osteoarthritic samples, this plot shows the 25%, 50% and 75% percentile for each sample as scatter and the median over these values for each sample group as solid line. (B) Volcano plots for group comparison P vs c for raw data and four different normalizations (for details see Data processing – normalization).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Variability of changes in gene expression depending on the normalization method. Figure showing fold changes and associated P-values of the comparison of gene expression levels of peripheral and central osteoarthritic chondrocytes for 10 OA-relevant genes.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
(A) Effect of normalization on fold changes. The individual diagrams show log2(fold changes) obtained from data normalized as indicated on the axes. The figure shows that normalization has an effect on fold changes, yet overall the fold changes derived from various normalizations are well correlated to each other. (B) Effect of normalization on P-values. The individual diagrams show log10(P-values) obtained from data normalized as indicated on the axes. The figure shows that normalization has an important effect on derived P-values; for most pairs of normalization methods, the correlation of derived P-values is weak, i.e., many genes are assigned with significant P-values when data is normalized with one method, but not with another.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 3
(A) Effect of normalization on fold changes. The individual diagrams show log2(fold changes) obtained from data normalized as indicated on the axes. The figure shows that normalization has an effect on fold changes, yet overall the fold changes derived from various normalizations are well correlated to each other. (B) Effect of normalization on P-values. The individual diagrams show log10(P-values) obtained from data normalized as indicated on the axes. The figure shows that normalization has an important effect on derived P-values; for most pairs of normalization methods, the correlation of derived P-values is weak, i.e., many genes are assigned with significant P-values when data is normalized with one method, but not with another.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 4
(A) Effect of normalization on expression profiles of various housekeeping genes. Samples classified in four disease stages (normal cartilage 1–18, early osteoarthritic cartilage 19–38, peripheral late stage 39–59 and central late stage 60–78) are listed on the X-axes. Red dots indicate the expression value (on the Y-axes) for a given sample (median expression value over spots representing one gene); the blue line indicates the median expression level over all samples belonging to one disease class. The individual diagrams show distinct housekeeping genes (columns) and normalizations (rows). The plot shows that most so-called housekeeping genes show significant variations in their expression levels between different disease stages, and normalization has an important effect on the expression profiles of these genes (∗the values completely outside off the usual range presumably are due to measurement errors). (B) Demonstration of the variation in expression levels of a panel of housekeeping genes (Applied Biosystems ‘Human endogenous control plate’) in three stimulation experiments as measured with the high-precision TAQMAN device. Y-axis: difference in number of PCR rounds, corresponding to log2ratio of expression. RPS18: ribosomal protein S18; RPLP0: ribosomal protein, large, P0; ACTB; PPIA: peptidylprolyl isomerase A (cyclophilin A); GAPDH; PGK1: phosphoglycerate kinase 1; B2M: beta-2-microglobulin; GUSB: glucuronidase, beta; TFRC: transferrin receptor.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions