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Table 1 Characteristics of different methylation analysis techniques and reported recommendations for assay design

From: Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome

 

Pyrosequencing

MSP

MS-HRM

Material

FF preferred

FFPE/FF

FF preferred

Closed-tube

  

€/sample

4.76

1.21–1.64b

1.76

Amplicon length (bp)

<400a/<200 bp

<160

<200

Annealing temp. (°C)

50–68

56a/60–66

58–66

Number of CpG dinucleotides included in primer

1 or less/none

1 or lessa/3 or more

2 or less

Analysis of CpG dinucleotides

CpG dinucleotides in between primers

CpG dinucleotides covered by primers

CpG dinucleotides covered and in between primers

  1. MSP methylation-specific PCR, MS-HRM methylation-sensitive high-resolution melting, FFPE formalin-fixed paraffin-embedded, FF fresh frozen
  2. aConditions of preamplification PCR (in case of nested-MSP: flank primers)
  3. bDirect-MSP and nested-MSP