Table 2. Tissue expression pattern of genes associated with TDMs: quantitative RT-PCR analysis.
| PCR, ΔCt ± SD
|
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|---|---|---|---|---|---|---|---|---|---|---|---|
| Locus | TDM location | Methyl. (tissue) | Gene | RT-PCR method | Brain | Kid | Liv | Col | Tes | Mus | GNF expression |
| Pst3 | 5′ promoter | U (testis) | Ddx4 | SYBR-G | 13.5 ± 0.6 | 14.6 ± 1.5 | 18.2 ± 1.7 | 14.6 ± 0.7 | -1.5 ± 0.3 | 17.4 ± 1.4 | Testis only/high expression |
| Pst6 | 3′ exon | U (testis) | Hsp-1-like | SYBR-G | 9.4 ± 1.4 | 7.6 ± 1.6 | 8.6 ± 1.2 | 5.9 ± 0.9 | -0.9 ± 0.1 | 10.7 ± 0.5 | Testis only/high expression |
| Pst6 | 3′ exon | U (testis) | Hsp1-like | TaqMan | 8.9 ± 1.3 | 10 ± 0.3 | 12.8 ± 1.1 | 10.7 ± 0.9 | -1.3 ± 1.1 | 6.7 ± 3.6 | Testis only/high expression |
| Pvu29 | 5′ promoter | U (testis) | RIKEN cDNA | SYBR-G | 7.2 ± 0.2 | 9.1 ± 0.4 | 8.8 ± 0.6 | 8.9 ± 0.5 | 2.7 ± 0.7 | 10.9 ± 0.3 | Broad pattern/high expression in testis |
| Pst61 | 5′ promoter | M (liver) | Gata2 | SYBR-G | 9.7 ± 0.4 | 7.5 ± 1.1 | 12.4 ± 0.9 | 11.1 ± 1.1 | 5.6 ± 0.4 | 10.2 ± 0.1 | Broad pattern/low expression in liver |
| Pst61 | 5′ promoter | M (liver) | Gata2 | TaqMan | 3.9 ± 1.5 | 1.6 ± 0.3 | 9.6 ± 1.8 | 6.4 ± 0.9 | 9.1 ± 0.6 | 7.6 ± 3.5 | Broad pattern/low expression in liver |
| Pvu2 | 3′ exon | U (testis) | Mct-like | SYBR-G | 8.4 ± 0.8 | 7.4 ± 0.4 | 8.1 ± 1.4 | 4.2 ± 0.9 | 7.5 ± 0.7 | 11.3 ± 1.6 | ND |
| Pvu66 | 3′ exon | M (liver) | Adra1b | SYBR-G | 8.1 ± 0.3 | 10.9 ± 1.1 | 6.9 ± 1.5 | 12.5 ± 2.5 | 11.8 ± 0.8 | 12.9 ± 5.7 | Broad pattern |
Genes associated with TDMs were identified by blat by using the University of California, Santa Cruz, Genome Bioinformatics database (http://genome.ucsc.edu). The methylation (methyl.; U, unmethylated; M, methylated) state (inferred by RLGS, MSP, and/or bisulfite sequencing) and location of the TDM relative to the gene are indicated. Although Pst6 is located in a 3′ exon, a CpG island 500 bp upstream of the promoter for Hspa11 had a methylation status similar to Pst6 (data not shown). Quantitative, real-time PCR was performed by using SYBR Green (SYBR-G) and TaqMan probes (see Methods). Expression levels were standardized to GAPDH by calculating ΔCt (ΔCt = Gene Ct – GAPDH Ct). The lower the ΔCt, the higher the mRNA level. ΔCt values with the highest mRNA level are shown in bold, and ΔCt values with the lowest mRNA level are shown in italics. A difference of one cycle is equivalent to an ≈2-fold difference in mRNA. Also indicated is the expression pattern determined from the GNF gene expression database (http://symatlas.gnf.org/SymAtlas). Kid, Kidney; Liv, liver; Col, colon; Tes, testis; Mus, muscle.