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Open Access Research article

Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cells

Lihan Zhou12, Qing-En Lim1, Guoqiang Wan12 and Heng-Phon Too12*

Author Affiliations

1 Department of Biochemistry, National University of Singapore, 119260, Singapore

2 Chemical Pharmaceutical Engineering, Singapore-Massachusetts Institute of Technology Alliance, 117576, Singapore

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BMC Genomics 2010, 11:75  doi:10.1186/1471-2164-11-75

Published: 29 January 2010

Additional files

Additional file 1:

RT-qPCR assay design and performance. Efficiencies of amplification and inter/intra-assay precisions of the assays used to measure the twenty candidate reference genes, two commonly used housekeeping genes and three target genes quantified in this study.

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Additional file 2:

Stability rankings of twenty candidate reference genes, ACTB and GAPDH in treatment and time-point subgroups. Stability rankings were determined by NormFinder (Italic) and geNorm, for each stimulus (Additional File 2) or time point (Additional File 2) subgroup. The top two candidate genes (RPL19 and RPL29) in overall ranking (Figure 4) were bolded in red and the two HKGs were bolded and highlighted in grey.

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Additional file 3:

Calculations of the deviation from NFtop2. Illustration of the calculation of the deviation of different normalization factors (NFRPL19/RPL29; NFACTB and NFGAPDH ) from NFtop2 (NFRPL10A/RPL29 for NGF group), in 12 control and 12 NGF treated samples.

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Additional file 4:

Time course analysis of GAPDH expression in NGF induced PC12 differentiation. A detailed time course analysis showing the up-regulation of GAPDH transcript expression by NGF treatment in PC12 cells, normalized by the geometric mean of RPL19 and RPL29.

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Additional file 5:

Normalized target gene expression regulation in PC12 cells differentiated with GDNF, Forskolin and Y27632. Fold changes in transcript expressions of Egr-1 (i), Integrin alpha 1, ITGA1 (ii), and Crystallin alpha b, CRYAB (iii), in GDNF-GFRa1a-RET9 (A), GDNF-GFRa1a-RET51 (B), Forskolin (C), Y27632 (D) treated samples relative to that of control were normalized by geometric mean of top 2 reference genes in each subgroup; geometric mean of RPL19/RPL29; ACTB or GAPDH. Normalization by ACTB resulted in the over-estimation of target gene expression. Normalization by GAPDH led to either under- or over-estimation of target gene expression. Dotted line represents the 2-fold difference between treatment and control subjects, a cut off commonly used to distinguish significant changes from insignificant ones. Significant differences between fold changes normalized by various reference gene(s) were calculated using the paired Student's t test. A value of p < 0.05 was considered significant (**p < 0.01; *p < 0.05)

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