Table II.
Respective Reference Genes and Cellular Genes Encoding RV Interaction Partners and Viral Genes With Respective Data for RT‐qPCR
| Gene | Amplicon size (bp) | ESlope (%)a | ELinReg (%)b | Ct | Ct of RT −vec | P (bp)c |
|---|---|---|---|---|---|---|
| Candidate reference genes | ||||||
| TBP | 226 | 86 | 84 | 25 | — | — |
| HPRT1 | 94 | 90 | 88 | 20 | — | — |
| PPIA | 325 | 96 | 73 | 20 | >36 | +(602) |
| HUEL | 104 | 94 | 86 | 24 | — | — |
| B2MG | 92 | 89 | 73 | 20 | >36 | — |
| β‐actin | 317 | 82 | 78 | 18 | >36 | +(506, 296) |
| GAPDH | 86 | 95 | 89 | 19 | 31 | +(67) |
| RPII | 632 | 95 | 82 | 28 | — | +(609) |
| ECHS | 200 | 89 | 85 | 22 | >36 | — |
| Genes possibly relevant for RV replication | ||||||
| p32 | 77 | n.d. | 90 | 19 | >36 | — |
| p53 | 77 | n.d. | 89 | 25 | 33 | — |
| RB | 76 | n.d. | 87 | 24 | >36 | — |
| p21 | 67 | n.d. | 87 | 27 | >36 | — |
| PABP | 96 | n.d. | 70 | 22 | 28 | — |
| SLC25A4 | 100 | n.d. | 87 | 21 | — | +(994) |
| SP100 | 110 | n.d. | 82 | 19 | — | +(1129) |
| Target on RV genome | ||||||
| P150 | 90 | n.d. | 84 | variable | >36 | — |
n.d., not determined; Ct, cycle threshold; RT −ve, minus RT control.
10‐fold serial dilutions of cDNA obtained from Vero cells were plotted against dilution factors. RT‐qPCR efficiencies (E) were calculated by the following equation (Rasmussen, 2001): E = 10(−1/slope). Only Ct values < 40 were included.
PCR efficiency was calculated based on the starting point of the exponential phase of amplification using LinReg PCR program.
The Ct values of RT −ve samples appear to be due to retropseudogenes (P) that lead to amplification of contaminating genomic DNA and could thus possibly interfere with RT‐qPCR results. If present, melting peaks for the RT−ve samples were distinguishable from the specific amplicon. The possible amplification of retropseudogenes with the primer sequences used in this study was determined by BLAT search [Kent, 2002. −, no retropseudogenes; +, retropseudogenes present, yielding amplicons of the indicated length.