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Acta Medica Martiniana
Volume 24 (2024): Issue 1 (April 2024)
Open Access
Optimizing Droplet Digital PCR Assay for Precise Assessment of MEIS1 Gene Promoter Methylation
Samec Marek
Samec Marek
,
Baranova Ivana
Baranova Ivana
,
Zavhorodnia Iryna
Zavhorodnia Iryna
,
Pec Martin
Pec Martin
,
Pecova Renata
Pecova Renata
and
Lucansky Vincent
Lucansky Vincent
| Apr 27, 2024
Acta Medica Martiniana
Volume 24 (2024): Issue 1 (April 2024)
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Published Online:
Apr 27, 2024
Page range:
21 - 28
Received:
Feb 19, 2024
Accepted:
Mar 11, 2024
DOI:
https://doi.org/10.2478/acm-2024-0004
Keywords
methylation
,
ddPCR
,
primer design
© 2024 Samec Marek et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Fig. 1
Promoter region of MEIS1 with marked TSS
Fig. 2
CpG island prediction generated by MethPrimer software
Fig. 3
PCR optimization. Figure 3A shows a positive signal generated by methylated probes using methylated control DNA (column B01); Figure 3B shows a positive signal generated by unmethylated probes using unmethylated control DNA (column B04). The positive signal generated by unmethylated probes in line B02 demonstrates the cross-reactivity of unmethylated probes with methylated control DNA.
Fig. 4
Concentration gradient: A) methylated DNA control using the methylated probe. B) unmethylated DNA control using the unmethylated probe.
Primer and probe sequences for MEIS1 detection
Primers
Name
Sequence (5´→3´)
MEIS1_1 Forward
TGGGGAGAGAGTTTGTAGG
MEIS1_1 Reverse
ACACAAACACCACACACC
Probes
Name
Sequence
MEIS1_1 Methylated probe (FAM-BHQ1)
CGGTCGCGGGTTATTGTTTGC
MEIS1_1 Unmethylated probe (HEX-IBFQ)
TGGTGGTTGTGGGTTATTGTTTGTGT