Table 4s MIQE list
Sample/Template
instruction
details
Source
If cancer,was biopsy screened for adjacent
normal tissue?
LiquidN2/RNAlater/formalin
If using samples>6months old
Human genomic DNA and lambda DNA
fresh/frozen/formalin
Series of dilution of either human genomic DNA or lambda DNA were freshly
prepared each time
Human genomic DNA was extracted with EZ Spin Column Whole Blood Genome
DNA Isolation Kit (Cat.# SK1261,Sangon). Lambda DNA was purchased from Sangon
(Cat# SD0011).
Not applied for this study
Method of preservation
Storage
time
(if
appropriate)
Handling
Extraction method
TriZol/columns
RNA:DNA-free
Concentration
RNA:integrity
Inhibition-free
Intron-spanning primers/no RT control
Nanodrop/ribogreen/microfluidics
Microfluidics/3':5' assay
Method of testing
Assay optimisation/validation
Accession number
RefSeq XX_1234567
Amplicon details
exon location, amplicon size
Primer sequence
even if previously published
Probe sequence*
In silico
identify LNA or other substitutions
BLAST/Primer-BLAST/m-fold
-80 degree refrigerator
The genomic DNA samples were stored at -80 degree within 6 months
About 10ng/l
Not applied for this study
Not applied for this study
Amplifications were not designed for any specific gene or DNA fragment.
Primers were randomly designed.
Human genome amplification: B9-10 (862 bp amplicon), A99-100 (803 bp amplicon), B17-18 (994
bp amplicon), A93-94 (967 bp amplicon); Lambda DNA amplification: Q7-8 (116 bp amplicon)
Randomly designed primers using Primer Premier 5:
B9 5’-AGGTAAAGGGGACTTTGTCTTGC-3’
B10 5’-CATTCTGCATGATGCGGTTATTA-3’
A99 5’-ACTGGGAAACTGTGACTGCTGC-3’
A100 5’-GACCTAACGTGGGACATAGAACAA-3’
B17 5’-ATTCCTTGCCATAGCCAACAGTA-3’
B18 5’-CGAGATTTCCTGCCCTAATCTTT-3’
A93 5’-GAACAGTCCAACAGCACTGAGTAAA-3’
A94 5’-AGCAGTGGGCCTTCTGTAAAAT-3’
Q7 5’-GGCTTGGCTCTGCTAACAC-3’
Q8 5’-TCATTCAACACCCGCACTAT-3’
Not applied for this study
Not applied for this study
empirical
primer concentration/annealing temperature
Priming conditions
PCR efficiency
Linear dynamic range
Limits of detection
oligo-dT/random/combination/target-specific
dilution curve
spanning unknown targets
LOD detection/accurte quantification
Intra-assay variation
copy numbers not Cq
RT/PCR
Protocols
Reagents
Duplicate RT
NTC
NAC
Positive control
Data analysis
Specialist software
Statistical justification
Transparent,validated
normalisation
fig7s
detailed description, concentrations, volumes
supplier, Lot number
ΔCq
Cq & melt curves
ΔCq beginning:end of qPCR
inter-run calibrators
e.g., QBAsePlus
e.g., biological replicates
e.g., GeNorm summary
All primers (2uM) were added 0.8ul for 12 ul PCR. Annealing temperatures
were all taken as 56 degree.
Target specific priming
dilution curve
At least to 10-6 dilution of the stock solution (10ng/ul) of the template DNA
When the stock solution (10ng/ul) was diluted 106 fold and 0.5ul (~1х105
lambda DNA molecules, as in fig1 and fig2) was added into a 12-ul qPCR,
the full repeatability can be realized both for SYBR Green I- and EvaGreen
–based qPCR, but Evagreen performed better in general. The lowest
concentrations for LOD detection were not further detected.
Assay variation of EvaGreen –based qPCR is generally smaller than that of
SYBR Green I-based qPCR in this study. One representative demonstration
can be seen in the fig7s.
Not applied for this study
Thermal Cycler DiceTM Real Time System Software
Biological replicates. Each set of PCR was repeated 2-4 times.
Not applied for this study. This study didn’t quantify any gene. Only basic
qPCR performance, such as melting-curves and amplification plots, was
presented in order to demonstrate the enhancing effects of QDs for SYBR
Green I- and EvaGreen –based qPCR.
A
B
C
D
E
Fig 7s Intra-assay or inter-assay variation of EvaGreen –based qPCR is generally smaller than that of SYBR Green I-based qPCR in this study. A)
amplification plot (SYBR Green I, Cat#SY1020, Solarbio); B) melting curve (SYBR Green I); C) amplification plot (EVAGREEN, Cat#31000,Biotium);
D) melting curve (EVAGREEN); E) Repeatability assay of EvaGreen –based qPCR with 10-2,10-3,10-5,10-6,10-7 dilutions of the template DNA. SYBR
Green I-based qPCR had similar repeatability.
PCR conditions:
94 oC-3min-（94 oC-20s-56 oC-32s-72 oC-24s）*45—72 oC-3min.
TAKARA QPCR TP800
Primers Q3-4 (amplicon size 106bp)
Q3 5’-CTAATAAGCCGATAGATAGCCAC-3’
Q4 5’-TACCTTTCCGCCATAACTGT-3’
12ul qPCR system:
12 ul reaction
2 *PCR buffer (NPK02)
1 ul
6.0uL
ddH20
2.2uL
Dye (20х)
0.6uL
QDs
1.5 ul
Taq
0.2 ul
Human genomic DNA 0.5uL
Primer 3-4
(10ng/ul)
-1
Template dilution
0.5
0.5*10
Plot line color
red
Bright blue
0.5*10
green
-2
0.5*10
yellow
-3
0.5*10
pink
-4
0.5*10
blue
-5
0.5*10-6
2.7*10-7
orange
black