Validation of housekeeping genes for normalizing RNA expression in real-time PCR

Figures & data

Table 1.  Housekeeping Genes Selected for Group 1 and Group 2 Panels

Group 1 Housekeeping Genes	Group 2 Housekeeping Genes
Human acidic ribosomal protein (HuPO)	Human acidic ribosomal protein (HuPO)
β-Actin (BA)	Elongation factor-1-α (EF-1-α)
Cyclophylin (CYC)	Metastatic lymph node 51 (MLN51)
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)	Ubiquitin conjugating enzyme (UbcH5B)
Phosphoglycerokinase (PGK)
β2-Microglobulin (B2M)
β-Glucuronidase (GUS)
Hypoxanthine phosphoribosyltransferase (HPRT)
Transcription factor IID TATA binding protein (TBP)
Transferrin receptor (TfR)

Table 2.  Primer and Probe Sequences Used to Quantify Gene Expression by Real-Time PCR

Genes	Sequences	Product Size (bp)	R ^2
EF-1-α	probe: 5′-(FAM-AGCGCCGGCTATGCCCCTG-TAMRA)-3′	59	0.992
	primer 1: 5′-CTGAACCATCCAGGCCAAAT-3′
	primer 2: 5′-GCCGTGTGGCAATCCAAT-3′
MLN51	probe: 5′-(FAM-AGGCCTGTGGAAGCTGGTGGGC-TAMRA)-3′	72	0.999
	primer 1: 5′-CAAGGAAGGTCGTGCTGGTT-3′
	primer 2: 5′-ACCAGACCGGCCACCAT-3′
UbcH5B	probe: 5′-(FAM-TGATCTGGCACGGGACCCTCCA-TAMRA)-3′	64	0.999
	primer 1: 5′-TGAAGAGAATCCACAAGGAATTGA-3′
	primer 2: 5′-CAACAGGACCTGCTGAACACTG-3′
HuPO	Assays-on-Demand (Applied Biosystems)	110	0.999
Group 1	TaqMan Human Endogenous Control Plate (Applied Biosystems)	N.A.	N.A.

EF-1-α, elongation factor-1-α; MLN51, metastatic lymph node 51; UbcH5B, ubiquitin conjugating enzyme; HuPO, human acidic ribosomal protein; N.A., not applicable.

Figure 1.  Real-time PCR cycle threshold values in blood samples.

Expression levels of group 1 genes (A) and group 2 genes (B) are shown as medians (lines), 25th percentile to the 75th percentile (boxes) and ranges (whiskers) for 16 human blood samples (4 healthy and 4 tuberculosis patients at two time points 4 h apart). HuPO, human acidic ribosomal protein; BA, β-actin; CYC, cyclophylin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerokinase; B2M, β₂-microglobulin; GUS, β-glucuronidase; HPRT, hypoxanthine phosphoribosyltransferase; TBP, transcription factor IID TATA binding protein; TfR, transferrin receptorl; UbcH5B, ubiquitin conjugating enzyme; EF-1-α, elongation factor-1-α; MLN51, metastatic lymph node 51.

Figure 2.  Fold change in gene expression.

Variability of selected group 1 genes and group 2 genes in human whole blood shown as an average fold change from the mean (columns) and maximum fold change (error bars). HuPO, human acidic ribosomal protein; BA, β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxanthine phosphoribosyltransferase; B2M, β₂-microglobulin; EF-1-α, elongation factor-1-α; UbcH5B, ubiquitin conjugating enzyme.

Figure 3.  Real-time PCR cycle threshold values in peripheral blood mononuclear cell (PBMC) culture samples.

Expression levels of group 1 genes (A) and group 2 genes (B) are shown as medians (lines), 25th percentile to the 75th percentile (boxes), and ranges (whiskers) for 12 human PBMC culture samples (4 tuberculosis patients at 3 time points). HuPO, human acidic ribosomal protein; BA, β-actin; CYC, cyclophylin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerokinase; B2M, β₂-microglobulin; GUS, β-glucuronidase; HPRT, hypoxanthine phosphori-bosyltransferase; UbcH5B, ubiquitin conjugating enzyme; EF-1-α, elongation factor-1-α; MLN51, metastatic lymph node 51.

Figure 4.  Fold change in gene expression.

Variability of selected group 1 genes and group 2 genes in human peripheral blood mononuclear cell (PBMC) culture shown as an average fold change from the mean (columns) and maximum fold change (error bars). HuPO, human acidic ribosomal protein; BA, β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxanthine phosphoribosyltransferase; B2M, β₂-microglobulin; EF-1-α, elongation factor-1-α; UbcH5B, ubiquitin conjugating enzyme.