Quantitative Methylation Specific PCR (qMSP)   

Materials and Reagents

1. TaqMan^® Universal Master Mix II no UNG (Life Technologies, catalog number: 4440039 )
   
2. TaqMan MGB probes (custom synthesis) (Life Technologies)
   
3. EZ DNA Methylation-Gold^TM kit (ZymoResearch, catalog number: D5006 )
   
4. The methylation-specific primer and probe sequences (see Recipes)
   

Equipment

1. PCR cabinet
   
2. PCR plates
   
3. Centrifuge (Sigma-Aldrich)
   
4. PCR thermal cycler (Life Technologies/Applied Biosystems, model: 9700 )
   
5. Real time PCR machine (Life Technologies/Applied Biosystems, model: 7500 FAST )
   

Procedure

1. Primer/probe design
   Note: Primer/probe design is of major importance for the specificity of the reaction, i.e. to amplify only methylated bisulphite-converted target in the presence of excess unmethylated target. The primers should include 3-5 CG dinucleotides. The inclusion of at least two such CGs within the six 3’ end of the primer significantly increases specificity. This is in addition to the usual rules on primer stability, GC content and secondary structure avoidance apply in real time PCR assay design.
   
2. One μg DNA was converted by sodium bisulphite using the EZ DNA Methylation-Gold^TM kit and following the supplier’s protocol but eluting in 50 μl elution buffer (instead of the recommended 10 μl).
   
3. The qMSP reactions contained 1x TaqMan^® Universal Master Mix II non-UNG, 250 nM probe, 300-900 nM primers (Table 1) and 2 μl eluate from the bisulphite treated DNA sample.
   Note: The primer concentration is an important determinant of analytical sensitivity/specificity and has to be ascertained experimentally In other words the analytical sensitivity threshold is set to the dilution that has an overlapping 95% Confidence Interval with the unmethylated control reaction. In practical terms, the highest sensitivity one can use is the one that is always at least 2 ΔCt. lower than the unmethylated control.
   
   Table 1. Primer-probe concentrations for oligo mixes
   

   Primer/probe mix	Final concentration (nM)
   	Fwd primer	Rev Primer	Probe
   p16	700	700	250
   TERT	250	250	250
   RASSF1	700	700	250
   TMEFF2	900	900	250
   CYGB	300	300	250
   RARb	500	500	250
   DAPK1	250	250	250
   p73	250	250	250
   WT1	750	750	250
   CDH13	250	250	250
   ACTB	900	900	250



4. PCR plates were sealed and span at 4,000 x g for 1 min prior to be placed in the thermal cycler in order to bring all the reaction volume to the bottom and ensure removal of bubbles from the reaction mix.
   
5. The reactions were performed in duplicate on a 7500 FAST real time cycler under the following thermal profile: 95 °C for 10 min activation step followed by 50 cycles consisted of denaturation at 95 °C for 15 sec, annealing and extension at 58 °C–65 °C (Table 2, depending on the assay) for 1 min.
   
   Table 2. Annealing information for qMSP optimised conditions
   

   Genes	Annealing temp (°C)	Time (sec)
   p16	60	60
   RASSF1	60	60
   CYGB	64	5
   	61	55
   RARB	65	5
   	62	55
   TERT	65	5
   	62.5	55
   WT	62	60
   ACTB	58	20
   	60	40
   CDH13	64	5
   	61	55
   DAPK	65	5
   	62.5	55
   P73	65	5
   	62.5	55
   TMEFF	58	20
   	60	40

Recipes

1. The methylation-specific primer and probe sequences are listed in Table 3. In the initial steps of assay development it became apparent that probes bearing minor groove binding moiety (Taqman MGB probes) provided significantly higher assay specificity. In addition, due to their smaller size, they allow for a more flexible assay design.
   
   Table 3. Nucleotide sequences of methylation specific primers and probes for the qMSP assays utilised in the BW screening. The ACTB assay is methylation-independent acting as DNA input control.
   

   Primer/probe name	Sequence 5’ →3’	Modification
   p16meth_F	GGAGGGGGTTTTTTCGTTAGTATC
   p16meth_R	CTACCTACTCTCCCCCTCTCCG
   p16meth_P	AACGCACGCGATCC	FAM-MGB
   RASSF1meth_F	GTGGTGTTTTGCGGTCGTC
   RASSF1meth_R	AACTAAACGCGCTCTCGCA
   RASSF1_P	CGTTGTGGTCGTTCG	FAM-MGB
   TMEFF2meth_F	GGAGAGTTAAGGCGTTTCGTAGTTC
   TMEFF2meth_R	CGTGGGAAGAGGTAGTCGGG
   TMEFF2meth_P	GTTTTTAGTTCGTTCG	FAM-MGB
   TERTmeth_F	TTGGGAGTTCGGTTTGGTTTC
   TERTmeth_R	CACCCTAAAAACGCGAACGA
   TERTmeth_P	AGCGTAGTTGTTTCGG	FAM-MGB
   CYGBmeth_F	GTGTAATTTCGTCGTGGTTTGC
   CYGBmeth_R	CCGACAAAATAAAAACTACGCG
   CYGBmeth_P	TGGGCGGGCGGTAG	FAM-MGB
   RARbmeth_F	GATTGGGATGTCGAGAACGC
   RARbmeth_R	ACTTACAAAAAACCTTCCGAATACG
   RARbmeth_P	AGCGATTCGAGTAGGGT	FAM-MGB
   DAPK1meth_F	CGAGCGTCGCGTAGAATTC
   DAPK1meth_R	ACCCTACAAACGAACTAACGACG
   DAPK1meth_P	AGCGTCGGTTTGGTAG	FAM-MGB
   p73meth_F	TTGTTTTTTGGATTTTAAGCGTTTC
   p73meth_R	CACCCGAATCTCTCCTAACCG
   p73meth_P	TAACGCTAAACTCCTCG	FAM-MGB
   WT1meth_F	GAGGAGTTAGGAGGTTCGGTC
   WT1meth_R	CACCCCAACTACGAAAACG
   WT1meth_P	AGTTCGGTTAGGTAGC	FAM-MGB
   CDH13meth_F	CGTGTATGAATGAAAACGTCGTC
   CDH13meth_R	CACAAAACGAACGAAATTCTCG
   CDH13meth_P	CGTTTTTAGTCGGATAAAA	FAM-MGB
   ACTBmgb_F	GGGTGGTGATGGAGGAGGTT
   ACTBmgb_R	TAACCACCACCCAACACACAAT
   ACTBmgb_P	TGGATTGTGAATTTGTGTTTG	VIC-MGB

   
   Cycle threshold (Ct) values for each target were normalized for DNA input by calculating the ΔCt=Ct_(Target)-Ct_(ACTB). The values for al samples were transformed to relative quantity (RQ) compare to the calibrator (0.5% standard methylated DNA dilution) included in all experiments using the following type:
   RQ_sample = 2^-ΔΔCt, whereΔΔCt = ΔCt_sample - ΔCt_calibrator.
   Note: qMSP is a challenging version of real-time PCR and one needs to gain a very good understanding of the latter prior to engaging in qMSP experiments. The main additional challenge is the use of bisulphite DNA which is of lower quality but most importantly of lower complexity. This significantly affects the thermodynamic behavior of this template in the reaction. The authors are very happy to provide assistance to colleagues if needed; please email Dr. T Liloglou (tliloglo@liv.ac.uk).
   

Acknowledgments

This protocol is adapted from Nikolaidis et al. (2012).

References

1. Liloglou, T., Bediaga, N. G., Brown, B. R., Field, J. K. and Davies, M. P. (2012). Epigenetic biomarkers in lung cancer. Cancer Lett 342(2): 200-212.
   
2. Nikolaidis, G., Raji, O. Y., Markopoulou, S., Gosney, J. R., Bryan, J., Warburton, C., Walshaw, M., Sheard, J., Field, J. K. and Liloglou, T. (2012). DNA methylation biomarkers offer improved diagnostic efficiency in lung cancer. Cancer Res 72(22): 5692-5701.