Background
Results
Evaluation of candidate reference gene expression in unstimulated and influenza a stimulated PBMCs and T-cells
Symbol | Name | Accession Number | Amplicon Size (bp) | IDT Assay ID |
---|---|---|---|---|
ACTB | β-actin | NM_001101.5 | 110 | Hs.PT.39a.22214847 |
GAPDH | Glyercaldehyde-3-phostphate dehydrogenase | NM_002046.7 | 143 | Hs.PT.39a.22214836 |
RPL13a | Ribosomal protein, L13a | NM_012423.4 | 106 | Hs.PT.58.45725862 |
RPS18 | Ribosomal protein, S18 | NM_022551.3 | 130 | Hs.PT.58.14390640 |
SDHA | Succinate dehydrogenase complex flavoprotein subunit A | NM_004168.4 | 150 | Hs.PT.58.41017719 |
UBE2D2 | Ubiquitin-conjugating enzyme E2D2 | NM_181838.1 | 119 | Hs.PT.58.622887 |
Symbol | Primer and Probe Sequences (5′-3′) | Exons | Average Efficiency (%)a |
---|---|---|---|
ACTB | F: ACAGAGCCTCGCCTTTG R: CCTTGCACATGCCGGAG P: /5HEX/TCATCCATG/ZEN/GTGAGCTGGCGG/3IABkFQ/ | 1–2 | 99.5 |
GAPDH | F: ACATCGCTCAGACACCATG R: TGTAGTTGAGGTCAATGAAGGG P: /5HEX/AAGGTCGGA/ZEN/GTCAACGGATTTGGTC/3AIBkFQ/ | 2–3 | 96.8 |
RPL13a | F: GCCGCCCCTGTTTCAAG R: CTCGACCATCAAGCACCAG P: /5HEX/AGAAACCCT/ZEN/GCGACAAAACCTCCT/3IABkFQ/ | 1b-3 | 99.3 |
RPS18 | F: GTTCCAGCATATTTTGCGAGT R: GTCAATGTCTGCTTTCCTCAAC P: /5HEX/TCTTCGGCC/ZEN/CACACCCTTAATGG/3IABkFQ/ | 2–3 | 99.5 |
SDHA | F: TTTGATGCAGTGGTGGTAGG R: CAGAGCAGCATTGATTCCTC P: /5HEX/TGCAACAGT/ZEN/GTGTGACCTGGTAGG/3IABkFQ/ | 3–4 | 99.5 |
UBE2D2 | F: GTACTCTTGTCCATCTGTTCTCTG R: CCATTCCCGAGCTATTCTGTT P: /5HEX/CCGAGCAAT/ZEN/CTCAGGCACTAAAGGA/3IABkFQ/ | 6–8 | 99.3 |
Analysis of candidate reference genes using Silver’s method, geNorm, BestKeeper and NormFinder
ACTB | GAPDH | RPL13a | RPS18 | SDHA | UBE2D2 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Method | Measure | PBMC | T-cell | PBMC | T-cell | PBMC | T-cell | PBMC | T-cell | PBMC | T-cell | PBMC | T-cell |
Silver | σCq | 0.52 | 0.562 | 1.204 | 1.29 | 0.803 | 0.821 | 0.538 | 0.568 | 0.564 | 0.601 | 0.493 | 0.541 |
geNorm | M | 1.281 | 0.362 | 0.825 | 0.854 | 0.556 | 0.312 | 0.52 | 0.278 | 0.53 | 0.311 | 0.537 | 0.287 |
BestKeeper | SD | 0.29 | 0.20 | 0.84 | 0.96 | 0.52 | 0.43 | 0.25 | 0.33 | 0.26 | 0.29 | 0.22 | 0.35 |
r | 0.737 | 0.504 | 0.587 | 0.688 | 0.227 | −0.173 | 0.738 | 0.873 | 0.401 | 0.468 | 0.836 | 0.907 | |
NormFinder (Stability Value) | Intragroup (all samples) | 0.067 | 0.100 | 0.810 | 0.873 | 0.479 | 0.496 | 0.068 | 0.074 | 0.192 | 0.219 | 0.073 | 0.074 |
Intergroup (± virus) | 0.094 | 0.147 | 0.572 | 0.508 | 0.436 | 0.402 | 0.074 | 0.08 | 0.152 | 0.173 | 0.046 | 0.099 | |
Intergroup (Y/O) | 0.034 | 0.039 | 0.26 | 0.281 | 0.154 | 0.161 | 0.044 | 0.029 | 0.058 | 0.066 | 0.022 | 0.023 |