Additional file 5. MIQE checklist for authors, reviewers and editors
ITEM TO CHECK
EXPERIMENTAL DESIGN
Definition of experimental and control
Groups
Number within each group
Assay carried out by core lab or
Investigators’ lab?
Acknowledgement of authors'
contributions
SAMPLE
Description
Volume/mass of sample processed
Microdissection or macrodissection
Processing procedure
If frozen - how and how quickly?
If fixed - with what, how quickly?
Sample storage conditions and
duration
(especially for FFPE samples)
NUCLEIC ACID EXTRACTION
Procedure and/or instrumentation
IMPORTANCE
CHECKLIST
COMMENTS/WHERE?
E
N/A
E
D
N/A
YES
Investigators’ lab
D
YES
Authors’ contributions section
E
D
E
E
E
E
E
YES
YES
N/A
N/A
N/A
N/A
YES
Materials and Methods
Materials and Methods
E
N/A
DNA supplied by manufacturer
(materials and methods)
Name of kit and details of any
modifications
Source of additional reagents used
Details of DNase or RNAse treatment
Contamination assessment (DNA or
RNA)
Nucleic acid quantification
Instrument and method
Purity (A260/A280)
Yield
RNA integrity method/instrument
RIN/RQI or Cq of 3' and 5'
transcripts
Electrophoresis traces
Inhibition testing (Cq dilutions, spike
or other)
REVERSE TRANSCRIPTION
Complete reaction conditions
Amount of RNA and reaction
volume
Priming oligonucleotide (if using
GSP) and concentration
Reverse transcriptase and
concentration
Temperature and time
Manufacturer of reagents and
E
N/A
D
E
E
N/A
N/A
N/A
E
E
D
D
E
E
YES
YES
YES
N/A
N/A
N/A
D
E
N/A
YES
E
E
N/A
N/A
E
N/A
E
N/A
E
D
N/A
N/A
Materials and Methods
Materials and Methods
Materials and Methods
Available on request
Additional file 7
catalogue numbers
Cqs with and without RT
Storage conditions of cDNA
qPCR TARGET INFORMATION
Sequence accession number
D
D
N/A
N/A
E
YES
Location of amplicon
Amplicon length
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)
What splice variants are targeted?
qPCR OLIGONUCLEOTIDES
Primer sequences
RTPrimerDB Identification Number
Probe sequences
Location and identity of any
modifications
Manufacturer of oligonucleotides
Purification method
qPCR PROTOCOL
Complete reaction conditions
Reaction volume and amount of
cDNA/DNA
Primer, (probe), Mg++ and dNTP
concentrations
Polymerase identity and
concentration
D
E
E
YES
YES
YES
CDKN2A (p14ARF):
NC_000009.12
COL2A1: NC_000012.12
Additional file 6
Additional file 6
Available on request
D
YES
None detected by BLASTn
D
D
YES
N/A
Available on request
E
YES
Additional file 6
E
N/A
E
D
D
E
YES
N/A
YES
YES
Additional file 6
D
D
YES
YES
Materials and Methods
HPLC
E
E
YES
YES
Materials and Methods
Materials and Methods
E
YES
E
YES
Buffer/kit identity and manufacturer
Exact chemical constitution of the
buffer
Additives (SYBR Green I, DMSO,
etc.)
Manufacturer of plates/tubes and
catalog number
Complete thermocycling parameters
Reaction setup (manual/robotic)
Manufacturer of qPCR instrument
qPCR VALIDATION
Evidence of optimisation (from
gradients)
Specificity (gel, sequence, melt, or
digest)
E
D
YES
NO
Materials and Methods;
Manufacturer’s proprietary
AmpliTaq Gold® DNA
Polymerase; concentration is
Manufacturer’s proprietary
Materials and Methods
Manufacturer’s proprietary
E
N/A
D
YES
E
D
E
YES
YES
YES
D
YES
E
YES
Additional file 6
Additional file 6
Life Technologies 96-well
plates (Cat. no. 4306737)
Materials and Methods
Manual
Materials and Methods
Standard curves shown in
Additional file 7
Available on request
For SYBR Green I, Cq of the NTC
Standard curves with slope and yintercept
PCR efficiency calculated from
slope
Confidence interval for PCR
efficiency or standard error
R2 of standard curve
Linear dynamic range
Cq variation at lower limit
Confidence intervals throughout range
Evidence for limit of detection
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
E
N/A
YES
Additional file 7
E
YES
Additional file 7
D
N/A
E
E
E
D
E
E
YES
YES
YES
YES
YES
N/A
Additional file 7
Additional file 7
Additional file 7
Tables 1,2 and Figures 1,2
Additional file 7
E
YES
Materials and Methods
E
E
E
YES
YES
YES
Justification of number and choice of
reference genes
E
YES
Description of normalisation method
Number and concordance of
biological replicates
Number and stage (RT or qPCR) of
technical replicates
Repeatability (intra-assay variation)
Reproducibility (inter-assay variation,
%CV)
Power analysis
Statistical methods for result
significance
Software (source, version)
Cq or raw data submission using
RDML
E = Essential; D = Desirable
E
D
YES
N/A
Materials and Methods
Table 1, Figure 1 and Results
All NTCs showed no
amplification (as shown in
Additional file 7).
COL2A1 assay has been
previously described as an
effective methylation
independent reference control
gene for Methylight [40,56,57].
Materials and Methods
E
YES
Materials and Methods
E
D
YES
YES
Additional file 7
Standard deviation
measurements in Results
D
E
N/A
YES
E
D
YES
N/A
Materials and Methods and
Results
Materials and Methods