Table 1. PCR primers forward (F), reverse (R) and sequencing primers (S) used for the FOLR1 gene Pyrosequencing methylation assays.
| CpG site No. | Nucleotide No.* | Primer sequence, 5′-3′ | Ta (°C) | Size (bp) |
|---|---|---|---|---|
| 1 & 2 |
116, 146 |
F: GGGTTAGGATTGAGTTTTTTAATGTTTG |
59 |
93 |
| 5′CGI shore |
|
R: biotin- AACCCACCTACTCATACAACTT |
|
|
| |
|
S: TGAGTTTTTTAATGTTTGTATGAA |
|
|
| 3–5 |
712, 716, 748 |
F:GTTTTTTTAAAGTGTGGGATTATAGAAATG |
57.5 |
93 |
| 5′CGI shore |
|
R: biotin-AAAAACACAACCCAAAATTTTAC |
|
|
| |
|
S: GTGGGATTATAGAAATGAG |
|
|
|
6−8
|
2508, 2518, 2524
|
F:GGTTAGGATGGTTTTGATTTTTTAGTT |
52.9 |
263 |
|
CGI
|
|
R: biotin-ACCCCAAACTAAATACAATAACTTACTT |
|
|
| |
|
S: AAAATGTTGGGATTATAGG |
|
|
| 9–11 |
3457, 3466, 3477 |
F: GTTGGGATTTTTGAATTTGAGTTT |
61 |
125 |
| 3′CGI shore |
|
R: biotin-TCTTCCCACCATTACTCACAA |
|
|
| |
|
S: TGTATTAAAATTATTTAGGTGGAT |
|
|
| 12–14 |
3843, 3855, 3875 |
F:AGTGGGAGTTGTTTGTTAATTT |
59 |
199 |
| 3′CGI shore |
|
R: biotin-AACCCCACTCATAACTACAACATA |
|
|
| |
|
S: ATTTTTATAAGGTTAGTAATTATAG |
|
|
*
Nucleotide number is relative to the ATG start site.