Table 4s MIQE list
Sample/Template / instruction / detailsSource / Ifcancer,wasbiopsyscreenedforadjacentnormaltissue? / Human genomic DNA and lambda DNA
Methodofpreservation / LiquidN2/RNAlater/formalin / -80 degree refrigerator
Storagetime(ifappropriate) / Ifusingsamples>6monthsold / The genomic DNA samples were stored at -80 degree within 6months
Handling / fresh/frozen/formalin / Series of dilution of either human genomic DNA or lambda DNA were freshly prepared each time
Extractionmethod / TriZol/columns / 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).
RNA:DNA-free / Intron-spanning primers/no RT control / Not applied for this study
Concentration / Nanodrop/ribogreen/microfluidics / About 10ng/l
RNA:integrity / Microfluidics/3':5' assay / Not applied for this study
Inhibition-free / Method of testing / Not applied for this study
Assayoptimisation/validation
Accessionnumber / RefSeq XX_1234567 / Amplifications were not designed for any specific gene or DNA fragment. Primers were randomly designed.
Amplicondetails / exon location, amplicon size / Human genome amplification: B9-10(862bp amplicon), A99-100(803bp amplicon), B17-18(994bp amplicon), A93-94 (967 bp amplicon); Lambda DNA amplification: Q7-8 (116 bp amplicon)
Primersequence / even if previously published / 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’
Probesequence* / identify LNA or other substitutions / Not applied for this study
Insilico / BLAST/Primer-BLAST/m-fold / Not applied for this study
empirical / primer concentration/annealing temperature / All primers (2uM) were added 0.8ul for 12 ul PCR. Annealing temperatures were all taken as 56 degree.
Primingconditions / oligo-dT/random/combination/target-specific / Target specific priming
PCRefficiency / dilution curve / dilution curve
Lineardynamicrange / spanning unknown targets / At least to 10-6 dilution of the stock solution (10ng/ul) of the template DNA
Limitsofdetection / LOD detection/accurte quantification / 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.
Intra-assayvariation / copy numbers not Cq / Assayvariation 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.
RT/PCR
Protocols / detailed description, concentrations, volumes / Not applied for this study
Reagents / supplier, Lot number
DuplicateRT / ΔCq
NTC / Cq & melt curves
NAC / ΔCq beginning:end of qPCR
Positivecontrol / inter-run calibrators
Dataanalysis
Specialistsoftware / e.g., QBAsePlus / Thermal Cycler DiceTM Real Time System Software
Statisticaljustification / e.g., biological replicates / Biological replicates. Each set of PCR was repeated 2-4 times.
Transparent,validatednormalisation / e.g., GeNorm summary / 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.
fig7s
A
B
C
D
E
Fig 7sIntra-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
QDs / 1 ul
2 *PCR buffer (NPK02) / 6.0uL
ddH20 / 2.2uL
Dye (20х) / 0.6uL
Primer 3-4 / 1.5 ul
Taq / 0.2 ul
Human genomic DNA (10ng/ul) / 0.5uL
Template dilution / 0.5 / 0.5*10-1 / 0.5*10-2 / 0.5*10-3 / 0.5*10-4 / 0.5*10-5 / 0.5*10-6 / 2.7*10-7
Plot line color / red / Bright blue / green / yellow / pink / blue / orange / black