Table 2.
Results of quantitative reverse transcription–real-time PCR across selected intergenic regions
| Locus | cDNA primera | Primer set | Normalized ΔCT value ± SDb |
Interpretation | ||
|---|---|---|---|---|---|---|
| ess1H164R | upf1Δ | ess1H164R upf1Δ | ||||
| PTP1-SSB1 | RH | 1 | 0.34 ± 0.56 | 2.45 ± 5.82 | 0.58 ± 0.88 | No aberrant transcripts |
| GUS1-RTF1 | RH | 1 | 0.40 ± 0.99 | 6.59 ± 3.93 | 3.68 ± 1.24 | |
| TAE1-RGD1 | RH | 1 | 11.79 ± 1.05 | 9.85 ± 0.39 | 68.95 ± 1.02 | Readthrough |
| GSS | 1 | 6.37 ± 0.75 | 3.74 ± 1.12 | 226.76 ± 0.66 | ||
| GSS | 2 | 24.20 ± 1.18 | 12.01 ± 1.56 | 1,166.80 ± 0.51 | ||
| SMK1-SEC8 | RH | 1 | 18.77 ± 1.03 | 4.54 ± 1.30 | 28.80 ± 1.03 | CUT |
| GSS | 1 | 2.36 ± 0.38 | ND | 9.63 ± 0.39 | ||
| GSS | 2 | ND | ND | ND | ||
| KTR3-FTH1 | RH | 1 | 27.54 ± 0.54 | 8.37 ± 1.63 | 58.42 ± 0.55 | CUT (reverse) and readthrough |
| GSS | 1 | 3.63 ± 0.76 | 6.61 ± 1.89 | 42.23 ± 0.89 | ||
| GSS | 2 | 2.64 ± 0.79 | 4.75 ± 1.65 | 24.30 ± 0.77 | ||
| RNQ1-FUS1 | RH | 1 | 92.41 ± 1.10 | 10.57 ± 2.79 | 192.45 ± 0.66 | CUT (reverse) and readthrough |
| GSS | 1 | 9.49 ± 0.60 | 5.19 ± 1.73 | 191.00 ± 0.69 | ||
| GSS | 2 | 77.35 ± 0.53 | 18.16 ± 1.30 | 1,103.86 ± 0.34 | ||
| TEF2-MUD1 | RH | 1 | 594.97 ± 1.08 | 3.69 ± 1.42 | 7,152.18 ± 4.05 | CUT (forward) and readthrough |
| GSS | 1 | 168.90 ± 0.93 | 5.17 ± 1.46 | 5,887.07 ± 1.18 | ||
| GSS | 2 | 435.54 ± 1.51 | 9.17 ± 1.66 | 9,079.10 ± 1.57 | ||
| AIM5-TAE1 | RH | 1 | 1.23 ± 1.42 | 2.59 ± 2.26 | 3.44 ± 0.95 | Readthrough |
| GSS | 1 | 0.37 ± 1.18 | 1.84 ± 4.36 | 12.29 ± 2.20 | ||
| GSS | 2 | 1.50 ± 0.97 | 6.53 ± 4.88 | 55.66 ± 2.07 | ||
| YPR114W-RGC1 | RH | 1 | 91.35 ± 0.33 | 4.07 ± 0.77 | 12,677.66 ± 1.31 | CUT (reverse) |
| GSS | 1 | 1.27 ± 0.56 | 2.19 ± 4.13 | 9.84 ± 0.84 | ||
| GSS | 2 | 1.05 ± 0.92 | 1.57 ± 6.66 | 1.26 ± 0.42 | ||
| HMG2-LEU3 | RH | 1 | 3.47 ± 0.41 | 5.68 ± 2.22 | 15.21 ± 1.20 | Possible (minor) readthrough |
| GSS | 1 | 0.84 ± 0.49 | 0.92 ± 0.67 | 3.18 ± 1.25 | ||
| GSS | 2 | 0.33 ± 0.49 | 1.85 ± 1.01 | 11.24 ± 0.14 | ||
| FIG4-PFA3 | RH | 1 | 1.84 ± 1.72 | 2.65 ± 1.13 | 9.18 ± 2.60 | CUT (forward) |
| GSS | 1 | 255.41 ± 0.80 | 142.35 ± 1.44 | 5,395.35 ± 0.94 | ||
| GSS | 2 | ND | ND | ND | ||
| SLM1-SHQ1 | RH | 1 | 34.86 ± 1.02 | 2.90 ± 4.49 | 66.48 ± 1.13 | CUT (forward) |
| GSS | 1 | 17.63 ± 1.34 | 3.25 ± 4.90 | 218.40 ± 1.58 | ||
| GSS | 2 | ND | ND | ND | ||
| YLR173W-IDP2 | RH | 1 | 1.69 ± 0.38 | 4.17 ± 3.13 | 5.00 ± 0.77 | Readthrough |
| GSS | 1 | 0.75 ± 0.58 | 3.39 ± 4.08 | 10.60 ± 0.96 | ||
| GSS | 2 | 1.85 ± 0.16 | 25.05 ± 3.96 | 105.85 ± 0.37 | ||
| SKM1-MSB4 | RH | 1 | 1.11 ± 1.02 | 21.61 ± 5.69 | 13.67 ± 0.52 | CUT (forward) revealed by upf1Δ |
| GSS | 1 | 0.81 ± 1.02 | 29.45 ± 3.36 | 13.79 ± 0.78 | ||
| GSS | 2 | ND | ND | ND | ||
RH, random hexamers; GSS, gene and strand specific.
All data are expressed as the ratio over the data for the wild type. Data have been normalized to SNR6 expression (to control for RNA and cDNA integrity and qRT-PCR efficiency) and to a region within the 5′ coding region of the upstream gene (to control for any potential changes in mRNA levels in the mutant backgrounds). See Materials and Methods for details. Primer set locations are approximately as depicted in Fig. 7. cDNA synthesis was primed using either random hexamers or gene-specific primers oriented so as to template only forward transcripts, as indicated. Random-hexamer-primed cDNAs typically resulted in stronger signals than cDNA made using a single gene-specific primer (i.e., using the same PCR primer sets). N.B., for the FIG4-PFA3 GSS primer set 1 samples, since WT readthrough was undetectable, we set the ΔCT(control) value to 37 cycles, which is average for samples with no readthrough. ND, none detected.