Published online Oct 15, 2022. doi: 10.4251/wjgo.v14.i10.2038
Peer-review started: July 20, 2022
First decision: August 20, 2022
Revised: August 21, 2022
Accepted: September 1, 2022
Article in press: September 1, 2022
Published online: October 15, 2022
Processing time: 86 Days and 1.9 Hours
Methylated ring finger protein 180 (RNF180) can be used as a potential biomarker for gastric cancer (GC) diagnosis.
Standard and sensitive of methylation detection methods for in plasma are urgently needed in clinical practice.
We aimed to use droplet digital polymerase chain reaction (ddPCR) to quantify the methylation level of the RNF180 gene. A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.
The primer and probe were designed and selected, the conversion time of bisulfite was optimized, the ddPCR system was adjusted by primer concentration, amplification temperature and amplification cycles, and the detection limit of ddPCR was determined.
The best conversion time for blood DNA was 2 h 10 min, and that for plasma DNA was 2 h 10 min and 2 h 30 min. The results of ddPCR were better when the amplification temperature was 56 °C and the number of amplification cycles was 50. Primer concentrations showed little effect on the assay outcome. Therefore, the primer concentration could be adjusted according to the reaction system and DNA input. The assay required at least 0.1 ng of input DNA.
In summary, a ddPCR assay was established to detect methylated RNF180, which is expected to be a new diagnostic biomarker for GC.
The standard procedure of ddPCR in clinical practice should be performed and evaluated.