Supplemental table 2: Amplification efficiencies, sensitivity and linearity of the IgH
RQ PCR
a) SYBR Green
ASO PCR
% of tumor
plasma cells
(established
by flow
cytometry)
AS0 primer
orientation
Slope
Error
ASO#01
8.4%
Downstream
-3.584
0.078
ASO#02
34.4%
Downstream
-3.656
0.105
ASO#03
3.6%
Downstream
-3.670
0.189
ASO#04
15.0%
Upstream
-3.526
0,077
ASO#07
5.2%
Upstream
-3.885
0,124
ASO#08
52.4%
Upstream
-3.692
0.084
ASO#09
10.0%
Upstream
-3.918
0.185
ASO WI
100% clonal
cells
Downstream
-3.634
0.110
1- 2.44E-04
( 21.9 - 35)
3.05E-05
AS0 primer
orientation
Slope
Error
Linearity
(Cp range)
Sensitivity limit*
Upstream
-3.408
0.118
Upstream
-3.291
0.084
Upstream
-3.271
0,048
Upstream
-3.377
0.078
Upstream
-3.369
0.070
b) hydrolysis probe
% of tumor
plasma cells
ASO PCR
(established
by flow
cytometry)
ASO#054.8%
JH3i
ASO#0620.8%
JH6i
ASO#0852.4%
JH4i
ASO#1030.0%
JH5i
ASOWI –
JH4i
100% clonal
cells
Linearity
(Cp range)
1 – 1.95E-03
(25.5-35.5)
1- 2.44E-04
(27.4-33)
1 - 1.95E-03
(26.6 – 32.2)
1- 2.44E-04
(25.8 – 35.4)
1 - 1.95E-03
(25.5-36.0)
1 - 1.56E-02
(29.9-38.3)
1 - 1.95E-03
(30.3 - 40)
1 - 1.95E-03
(32.3-41.6)
1 - 1.95E-03
(25.7-34.7)
1 - 1.95E-03
(25.8-36.7)
1 - 2.44E-04
(25.9-38.1)
1 - 2.44E-04
(22.7-34.9)
Sensitivity limit *
1.95E-03
2.44E-04
1.95E-03
2.44E-04
1.95E-03
1.95E-03
2.44E-04
1.95E-03
2.44E-04
2.44E-04
2.44E-04
3.05E-05
* dilution of bone marrow diagnosis sample for which at least one of the triplicate gave a
fluorescent signal.
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Efficiency determination
Slopes from the standard curve, permitted to determine the efficiencies of ASO PCR.
The best efficiency is achieved when doubling the PCR specific product at each cycle
resulting in an efficiency of 2 and a slope of -3.32 according to the equation
E=10-1/slope. Slopes between -4.0 and -3.2 which corresponds to an efficiency varying
between 89% and 100%, are considered as acceptable.
Upstream versus downstream ASO IgH SYBR Green quantitative
PCR ASO IgH PCRs were adapted for SYBR Green I detection system. Successful
amplifications were obtained using the Qiagen SYBR Green kit either with the
downstream oriented ASO primer for 4 ASO PCRs or with the upstream oriented
ASO primer for 4 other ASO PCRs (Supplemental table 2a). For SYBR Green I
optimized ASO PCR reactions, an acceptable PCR efficiency was obtained with
slopes ranging from -3.526 to -3.918. It has been reported that smaller amplicons
were more efficiently amplified. Howewer, we did not reach the same conclusion as
the efficiencies between ASO downstream (PCR product from about 250 bp) and
upstream strategies (PCR product about 80 bp) were comparable. Therefore, both
ASO primer orientations can be recommended.
ASO RQ PCR with SYBR Green versus consensus hydrolysis probes
approaches
For SYBR Green I detection, the slopes examined for 8 ASO PCRs ranged from 3.526 to -3.918, corresponding to a mean value of -3.704 with an error ≤ to 0.189
(Supplemental table 2a). For hydrolysis probe detection, the slope mean of 5 ASO
PCRs, was higher and attained -3.337 (ranging from -3.271 to -3.408) with an error ≤
to 0.118 (Supplemental table 2b). These results indicated that hydrolysis probe
detection format led to a better PCR efficiency.
For the MM ASO PCR, the sensitivity limit varied between 1.95E-03 and 2.44E-04
and was reduced compared to the WI cell line. The reduced sensitivity limit for ASO
#01, #03 and #07 was explained by a weak initial tumor load inferior to 10% of total
MNC as established by flow cytometry. For hydrolysis probe format, the sensitivity
limit reached 2.44E-04 except for the ASO #05 PCR which only attained 1.95E-03
because of the low tumor burden (4,8%) in the BM sample DNA from diagnosis. For
ASO #08 PCR analysed by both methods, the linearity and the sensitivity limit were
reduced with the SYBR Green I detection approach, therefore the hydrolysis probe
detection was advised and used for the residual tumor cells follow-up when possible.
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