Supplemental table 1: Optimization of ASO IgH RQ PCR.
ASO primer and SYBR Green for
ASO primer and consensus
IgH RQ PCR
hydrolysis probe for IgH RQ PCR
Amplification
Patients
ability with
Id.
Successful
an intronic
amplification
Annealing
JH primer
and
temperature
Non specific
amplification
b
detection (a)
Successful
amplification
Annealing
and
temperature
Non specific
amplification
b
detection (a)
#01
JH4i positive
YES (25)
66°C
0/6
ND
#02
JH6i positive
YES (22)
62°C
1/6
ND
#03
JH4i negative
YES (30)
64°C
1/5
ND
#04
JH5i negative
YES (26)
62°C
0/6
#05
JH3i positive
ND
YES (32)
62°C
1/7
#06
JH6i positive
ND
YES (26)
64°C
0/6
#07
JH6i negative
YES (25)
64°C
0/6
NO
#08
JH4i positive
YES (30)
68°C
0/6
YES (29)
65°C
0/6
#09
JH4i negative
YES (30)
64°C
0/5
NO
#10
JH5i positive
YES (26)
62°C
0/5
Wi
JH4i positive
YES (23)
62°C
1/5
ND
YES (22)
62°C
0/5
NO
a
Cp for diagnosis BM DNA
b
number of non patient related samples amplification detected on the total number of
non-related samples tested for specificity screening.
ND: not done
The major parameter influencing IgH quantitative PCR was the annealing
temperature since MgCl2 concentration was pre-optimized in the reaction mixes.
Annealing temperature was set to 60°C and increased by progressive steps of 2°C
each. The best conditions corresponded to the balance between an adequate
specificity and a sufficient sensitivity. To control the risk of SYBR Green I non-specific
dsDNA binding, we performed specificity tests as described in Materials and Methods
section (Supplemental information). In order to fulfil this criteria, stringent PCR
conditions were necessary with high annealing temperature varying from 62°C to
68°C.
2 ASO RQ PCR were developed for the patient #08 and the cell line Wi with either
SYBR Green I or hydrolysis probe detection system(s). In this two cases indicated in
bold, adequate specificity were obtained with both methods.