Download Supplementary file S2. Description of the experimental details of the

Supplementary file S2.
Description of the experimental details of the RT-qPCR analyses according to the
checklist of the MIQE guidelines.
The items in the checklist summarize the characteristics of the RT-qPCR analyses according
to Bustin et al. [Clin Chem (2009) 611-622] and indicate where the details are described in
the manuscript.
ITEM TO CHECK
IMPORTANCE
CHECKLIST
E
Yes
E
Yes
D
Yes
D
Yes
E
D
Yes
Yes
E
Yes
Processing procedure
E
Yes
If frozen – how and how
quickly?
If fixed – with what, how
quickly?
Samples storage conditions
and duration (esp. for FFPE
samples)
NUCLEIC ACID EXTRACTION
Procedure and/or
instrumentation
E
Yes
E
N/A
E
Yes
Materials and Methods: Tissue
samples.
E
Yes
Name of kit and details of any
modifications
E
Yes
Source of additional reagents
used
Details of DNase or RNase
treatment
Contamination assessment
(DNA or RNA)
Nucleic acid quantification
E
No
Materials and Methods: RNA
isolation and cDNA synthesis.
References are indicated.
Materials and Methods: RNA
isolation and cDNA synthesis.
“miRNeasy Mini Kit” (Qiagen,
Hilden, Germany) Cat. No.217004
-
E
Yes
Treatment was not used
E
Yes
E
Yes
Materials and Methods: RNA
isolation and cDNA synthesis
Materials and Methods: RNA
ERXPERIMENTAL DESIGN
Definition of experimental and
control groups
Number within each group
Assay carried out by core lab or
investigator´s lab?
Acknowledgement of authors´
contributions
SAMPLE
Description
Volume/mass of sample
processed
Microdissection or
macrodissection
WHERE IN THE MANUSCRIPT;
ADDITIONAL COMMENT
Materials and Methods: Patients
and tissue samples.
Materials and methods: Patients
and tissue samples. See also:
Number and concordance of
biological replicates.
All assays were performed in
investigator´s lab.
Contributors who do not meet the
authorship as defined by the journal
are listed in the Acknowledgement
section.
Materials and Methods.
Materials and Methods: RNA
isolation and cDNA synthesis
Materials and Methods: RNA
isolation and cDNA synthesis;
macrodissections with histological
verification.
Materials and Methods: Tissue
samples.
Materials and Methods: Tissue
samples.
-
isolation and cDNA synthesis
Instrument and method
E
Yes
Purity (A260/A280)
D
Yes
Yield
D
Yes
RNA integrity
method/instrument
E
Yes
RIN/RQI or Cq of 3`and
5`transcripts
E
Yes
Electrophresis traces
Inhibition testing (Cq dilutions,
spike or other)
D
E
No
Yes
E
Yes
Amount of RNA and reaction
volume
E
Yes
Priming oligonucleotide (if using
GSP) and concentration
E
Yes
Reverse transcriptase and
concentration
E
Yes
Temperature and time
E
Yes
Manufacturer and reagents and
catalogue numbers
D
Yes
Cqs with and without RT
D*
Yes
Storage conditions of cDNA
qPCR TARGET INFORMATION
If multiplex, efficiency and LOD
of each assay
Sequence accession number
D
Yes
Materials and Methods:
Quantitative real-time PCR.
Supporting Information
Supplementary file S1; table 1.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 1.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 3.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 1.
Materials and Methods:
Quantitative real-time PCR and
reference indicated there.
Supporting Information:
Supplementary file S1; table 2.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 1.
There were no Cqs < 40 in
reactions without RT.
cDNA stored at -20°C.
E
N/A
-
E
Yes
Location of amplicon
D
N/A
Amplicon length
E
N/A
Supporting Information:
Supplementary file S1; table 3.
Supporting Information:
Supplementary file S1; specificity
guaranteed by the manufacturer.
Supporting Information:
REVERSE TRANSCRIPTION
Complete reaction condition
Materials and Methods: RNA
isolation and cDNA synthesis
Materials and Methods: RNA
isolation and cDNA synthesis
Materials and Methods: RNA
isolation and cDNA synthesis
Materials and Methods: RNA
isolation and cDNA synthesis;
Bioanalyzer 2100 (Agilent).
RIN values given in Materials and
Methods: RNA isolation and cDNA
synthesis.
Dilution experiments were
performed; PCR efficiencies were
found 90%-98%; see also
Supporting Information:
Supplementary file S3. For all
clinical samples, identical isolation
procedures were performed.
In silico specificity screen
(BLAST, etc.)
Pseudogenes,
retropseudogenes or other
homologs?
Sequence alignment
Secondary structure analysis of
amplicon
Location of each primer by
exon or intron (if applicable)
Supplementary file S1; specificity
guaranteed by the manufacturer.
Specificity guaranteed by the
manufacturer.
Specificity guaranteed by the
manufacturer.
E
N/A
D
N/A
D
D
N/A
N/A
-
E
Yes
What splice variants are
targeted?
qPCR OLIGONUCLEOTIDES
Primer sequences
E
Yes
Use of mRNA specific UPL assays;
specificity guaranteed by the
manufacturer.
Supporting Information:
Supplementary file S1; table 3.
E
N/A
RT Primer DB Identification
Number
Probe sequences
Location and identity of any
modifications
Manufacture of oligonucleotides
D
N/A
D**
E
N/A
Yes
D
Yes
Purification method
qPCR PROTOCOL
Complete reaction conditions
D
N/A
E
Yes
Reaction volume and amount of
cDNA/DNA
E
Yes
Primer, (probe), Mg++ and
dNTP concentration
E
Yes
Polymerase identity and
concentration
E
Yes
Buffer/kit identity and
manufacture
E
No
Exact chemical constitution of
the buffer
Additives (SYBR Green I,
DMSO, ect.)
Manufacturer of plates/tubes
and catalog number
D
Yes
E
Yes
D
Yes
Complete thermo cycling
parameter
E
Yes
Supporting Information:
Supplementary file S1; table 3.
Supporting Information:
Supplementary file S3; table 3.
TIB MOLBIOL Syntheselabor
GmbH.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 4 and
5.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 4.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 4.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 4.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Supplementary file S1; table 4.
The manufacturer does not provide
this information.
Assays without additional additives.
Materials and Methods:
Quantitative real-time PCR;
Supporting Information:
Supplementary file S1; table 4.
Materials and Methods:
Quantitative real-time PCR.
Supporting Information:
Reaction setup
(manual/robotic)
Manufacturer of qPCR
instruments
Supplementary file S1; table 5.
Manual setup.
D
Yes
E
Yes
LightCycler 480; see also Materials
and Methods: Quantitative real-time
PCR.
D
Yes
Specificity (gel, sequence, melt,
or digest)
SYBR Green I, Cq of the NTC
E
Yes
E
Calibration curves with slope
and Y-intercept
E
Not
applicable
Yes
Kits from Roche Diagnostics
GmbH. Optimization guaranteed
by the manufacturer.
Specificity guaranteed by
manufacturer.
-
PCR efficiency calculated from
slope
E
Yes
Confidence interval PCR
efficiency or standard error
r2 of standard curve
D
Yes
E
No
Linear dynamic range
E
Yes
Cq variation at lowest
concentration of the linear
interval of calibration curves
Confidence intervals throughout
range
Evidence for limit of detection
E
Yes
D
No
-
E
Yes
If multiplex, efficiency and LOD
of each assay
DATA ANALYSIS
qPCR analysis program
(source, version)
Cq method determination
Outlier identification and
disposition
Results of NTCs
E
N/A
Supporting Information:
Supplementary file S3.
Measurements of all were in linear
dynamic range. Thus, it was not
necessary to determine the LOD.
-
E
Yes
E
E
Yes
Yes
E
Yes
Justification of number and
choice of reference genes
E
Yes
Description of normalization
method
Number and concordance of
biological replicates
E
Yes
D
Yes
Number and stage (RT or
qPCR) of technical replicates
E
Yes
Repeatability (intra-assay
E
Yes
qPCR VALIDATION
Evidence of optimization (from
gradients)
Material and Methods: Quantitative
real-time PCR; see also Supporting
Information: Supplementary file S3.
Material and Methods: Quantitative
real-time PCR; see also Supporting
Information: Supplementary file S3.
Supporting Information:
Supplementary file S3.
Not provided by the LightCycler 480
software.
Supporting Information:
Supplementary file S3.
Supporting Information:
Supplementary file S3.
Materials and Methods:
Quantitative real-time PCR.
Cq >37 was decided as limit.
There were no outliers.
NTC did not result in any
amplification.
PBGD was used as reference
gene. Supporting Information,
Supplementary file S1; Reference
genes.
Materials and Methods: Data
analysis; GenEx v. 4.3.7.
Legend of figure 3: adjacent
nonmalignant samples n = 50;
tumor samples n = 50.
Materials and Methods:
Quantitative real-time PCR;
triplicate measurements.
Materials and Methods:
variation, %CV)
Reproducibility (inter-assay
variation, %CV)
Power analysis
Statistical methods for result
significance
D
N/A
D
E
No
Yes
Software (source, version)
E
Yes
Cq or raw data submission
RDML
D
No
Supplementary file S1; RT-qPCR.
Materials and Methods: Data
analysis. Results and Figure
legends.
Materials and Methods: Data
analysis.
-
E, essential information that must be submitted with the manuscript; D, desirable information that
should be submitted with the manuscript if available.