Background
Results
Stability of reference gene expression
Candidate Reference Genes | |||||
---|---|---|---|---|---|
Symbol | Name | Function | Assay ID& | r2 | E |
ACTB | Actin, beta | Structural protein | Hs99999903_m1 | 0.997 | 1.97 |
B2M | Beta-2-microglobulin | Beta chain of MHC I | Hs99999907_m1 | 0.999 | 2.00 |
CTBP1 | C-terminal binding protein 1 | Involved in cellular proliferation. | Hs00179922_m1 | 0.998 | 2.09 |
GAPDH | Glyceraldehydes-3-phosphate dehydrogenase | Glycolysis and gluconeogenesis | Hs99999905_m1 | 0.998 | 2.00 |
GOLGA1 | Golgi autoantigen, golgin subfamiliy A, 1 | Unknown function | Hs00608118_m1 | 1.000 | 1.96 |
PGK1 | Phosphoglycerate kinase 1 | Glycolysis | Hs99999906_m1 | 0.997 | 2.07 |
PPIA | Cyclophilin A (Peptidylprolyl isomerase A) | Protein folding | Hs99999904_m1 | 0.996 | 2.08 |
PPIB | Cyclophilin B (Peptidylprolyl isomerase B) | Associated with the secretory pathway | Hs00168719_m1 | 0.996 | 2.00 |
SDHA | Succinate dehydrogenase complex, subunit A | Electron transporter | Hs00417200_m1 | 0.998 | 2.10 |
TMBIM4 | Transmembrane BAX inhibitor motif containing 4 | Involved in apoptosis | Hs00211390_m1 | 1.000 | 2.00 |
TBP | TATA box binding protein | Transcription factor | Hs99999910_m1 | 0.999 | 2.10 |
UBC | Ubiquitin C | Protein degradation | Hs00824723_m1 | 0.999 | 2.05 |
Target Genes
| |||||
Symbol
|
Name
|
Function
|
Assay ID
|
r
2
|
Efficiency*
|
IL10 | Interleukin 10 | Antiinflammatory cytokine | Hs00174086_m1 | 0.989 | 2.01 |
TNF-α | Tumor necrosis factor | Proinflammatory cytokine | Hs00174128_m1 | 0.997 | 2.08 |
Candidate Reference Genes | ||||||
---|---|---|---|---|---|---|
GM [Cq] | AM [Cq] | Min [Cq] | Max [Cq] | SD [± Cq] | CV [% Cq] | |
ACTB | 23.26 | 23.30 | 21.05 | 28.52 | 0.92 | 3.95 |
B2M | 21.55 | 21.55 | 20.62 | 22.92 | 0.40 | 1.87 |
CTBP1 | 30.80 | 30.84 | 27.91 | 36.19 | 1.25 | 4.06 |
GAPDH | 23.89 | 23.91 | 22.87 | 26.86 | 0.58 | 2.43 |
GOLGA1 | 33.11 | 33.14 | 30.19 | 36.67 | 0.99 | 3.00 |
PGK1 | 26.08 | 26.09 | 24.08 | 27.66 | 0.57 | 2.17 |
PPIA | 23.70 | 23.70 | 23.02 | 24.50 | 0.22 | 0.91 |
PPIB | 26.45 | 26.45 | 25.28 | 27.95 | 0.37 | 1.40 |
SDHA | 25.61 | 25.63 | 24.33 | 29.06 | 0.80 | 3.11 |
TBP | 30.35 | 30.36 | 28.93 | 33.11 | 0.66 | 2.18 |
TMBIM4 | 29.25 | 29.27 | 27.23 | 31.16 | 0.80 | 2.74 |
UBC | 26.68 | 26.68 | 25.74 | 28.09 | 0.47 | 1.76 |
Target genes
| ||||||
GM [C
q
]
|
AM [C
q
]
|
Min [C
q
]
|
Max [C
q
]
|
SD [± C
q
]
|
CV [%C
q
]
| |
IL10 | 31.16 | 31.27 | 27.98 | 36.68 | 2.26 | 7.24 |
TNF-α | 26.68 | 26.89 | 22.52 | 31.80 | 3.28 | 12.21 |
i) geNorm analysis
ii) Normfinder analysis
Gene name | Stability value |
---|---|
ACTB | 0.036 |
PPIB | 0.044 |
GOLGA1 | 0.060 |
PGK1 | 0.072 |
CTBP1 | 0.076 |
TMBIM4 | 0.077 |
PPIA | 0.080 |
SDHA | 0.091 |
TBP*
|
0.100
|
B2M*
|
0.104
|
GAPDH | 0.118 |
UBC | 0.168 |
iii) Bestkeeper analysis
BestKeeper index vs. | GAPDH | ACTB | B2M | TMBIM4 | GOLGA1 | SDHA | TBP | PGK1 | PPIA | PPIB |
---|---|---|---|---|---|---|---|---|---|---|
Coefficient of correlation [r] | 0.73 | 0.96 | 0.80 | 0.77 | 0.93 | 0.79 | 0.80 | 0.65 | 0.70 | 0.88 |
p-value | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 | 0.001 |
Evaluation of different normalization strategies
TNF-α mRNA | ||||||||
---|---|---|---|---|---|---|---|---|
ACTB | GAPDH | gN comb | NF comb | |||||
fold change
|
p-value
|
fold change
|
p-value
|
fold change
|
p-value
|
fold change
|
p-value
| |
Nm LPS stim/non-stim | 172 | < 0.05 | 315 | < 0.05 | 104 | < 0.05 | 158 | < 0.05 |
Ec LPS stim/non-stim | 480 | < 0.05 | 596 | < 0.05 | 192 | < 0.05 | 288 | < 0.05 |
IL10 mRNA
| ||||||||
ACTB
|
GAPDH
|
gN comb
|
NF comb
| |||||
fold change
|
p-value
|
fold change
|
p-value
|
fold change
|
p-value
|
fold change
|
p-value
| |
Nm LPS stim/non-stim | 9 | < 0.05 | 16 | < 0.05 | 6 | < 0.05 | 8 | < 0.05 |
Ec LPS stim/non-stim | 13 | < 0.05 | 17 | < 0.05 | 6 | < 0.05 | 7 | < 0.05 |