Table 1.
Summary of primers detecting rGPx-3 mRNA and endogenous GPx-3.
| Primer Name | Sequence | Position based on Accession # AY310878 | Size |
|---|---|---|---|
| GPx-3 cDNA | |||
| GPx-3 cDNA NheI Forward | 5′ ATA TGC TAG C CC ATG GCC CGG CTG CTG CAG G 3′ | 2181 | 693 bp |
| GPx-3 cDNA XhoI Reverse | 5′ CCT ACT CGA G CC TTC CTC TTG ACC CCC AGG GCT 3′ | 9656 | |
| 100 bp 3′ UTR SECIS-containing fragment | |||
| GPx-3 SECIS_100 PmeI Forward | 5′ ATA TGT TTA AAC GGA CCC CAT GGC AGG GGT 3′ | 10296 | 125 bp |
| GPx-3 SECIS_100 PmeI Reverse | 5′ CCT AGT TTA AAC TTA GCC TGA ATG CAC TAA 3′ | 10379 | |
| 500 bp 3′ UTR SECIS-containing fragment | |||
| GPx-3 SECIS_500 PmeI Forward | 5′ ATAT GT TTA AAC T GCC TAC AGG TAT GCG TGA TTG 3′ | 9959 | 529 bp |
| GPx-3 SECIS_500 PmeI Reverse | 5′ CCT A GT TTA AAC AAG CCA GTG GAC CAG TGA GGG GTG A 3′ | 10476 | |
| Full Length 3′ UTR SECIS-containing fragment | |||
| GPx-3 SECIS Full Length PmeI Forward | 5′ ATA TGT TTA AAC CTG AAG GCC GTC TCA TCC 3′ | 9660 | 872 bp |
| GPx-3 SECIS Full Length PmeI Reverse | 5′ CCT AGT TTA AAC CTG CAG AAA GGC TTT TAC 3′ | 10515 | |
| GPx-3 cDNA Selenocysteine Mutant | |||
| SeCysMut Forward | 5′ CAA CGT GGC CAG CTA CTG TGG CCT GAC GGG CCA G 3′ | 6982 | Sec→Cys |
| SeCysMut Reverse | 5′ CTG GCC CGT CAG GCC ACA GTA GCT GGC CAC GTT G 3′ | 7015 | |
| Recombinant GPx-3 Transfects Detection | |||
| GPx3 His-tag Forward | 5′ GGT CAT CAT CAC CAT CAC CAT TGA 3′ | pcDNA 4/V5 His C vector 1053 | 95 bp (p100), 428 bp (p500), |
| SECIS Reverse | 5′ AGG TCC GCC CAC AAG GGC TTT G 3′ | 10358 | 731 bp (p800) |
| Recombiant GPx-3 Transfectants Detection with Real Time PCR | |||
| Real Time GPx-3 Forward | 5′ GCA ACG TCA AGA TGG ACA TCC T 3′ | 9592 | 112 bp |
| Real Time GPx-3 Reverse | 5′ GGA GAG GGT TAG GGA TAG GCT TAC 3′ | pcDNA 4/V5 His C vector 1029 | |
| TaqMan GPx-3 Probe | 5′ AGG TCA CCC ATT CGA A 3′ | pcDNA 4/V5 His C vector 989 | |
| Endogenous GPx-3 Detection | |||
| Endogenous 5′UTR GPx-3 Forward | 5′ ATA TCA CCC CGC CAT GGC CCG GCT G 3′ | Position 56 based on Bierl et al JBC 2004 | 688 bp |
| Endogenous GPx-3 cDNA Reverse | 5′ CC TTC CTC TTG ACC CCC AGG GCT 3′ | 9656 |
Restriction enzymes sites for NheI, XhoI, and PmeI are italicized preceded by 4 bp overhangs.
Endogenous 5′UTR GPx-3 Forward also contains 4 bp overhangs.
The Cys codon in the rGPx-3 Cys mutant is highlighted in bold and underlined.