Additional file 8. Digital 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?
Power analysis
SAMPLE
Description
Volume or 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
Quantification - instrument/method
IMPORTANCE CHECKLIST
COMMENTS/WHERE?
E
N/A
E
D
N/A
YES
D
N/A
E
E
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
Storage conditions: temperature, concentration,
duration, buffer
DNA or RNA quantification
Quality/integrity-instrument/method; e.g.
RIN/RQI and trace or 3’:5’
Template structural information
Template modification (digestion, sonication,
pre-amplification etc.)
Template treatment (initial heating or chemical
denaturation)
Inhibition dilution or spike
E
YES
DNA supplied by
manufacturer (materials
and methods)
Materials and Methods
E
E
YES
N/A
E
E
N/A
YES
Materials and Methods
E
YES
Materials and Methods
E
YES
DNA samples were diluted
after RE digestion and
bisulfite treatment as
described in Materials and
Methods in order to reduce
effects of potential
inhibitors
DNA contamination assessment of RNA sample
Details of DNase treatment where performed
Manufacturer of reagents used and catalogue
number
Storage of nucleic acid: temperature,
concentration, duration, buffer
REVERSE TRANSCRIPTION (If necessary)
cDNA priming method + concentration
One or two step protocol
Amount of RNA used per reaction
Detailed reaction components and conditions
RT efficiency
Estimated copies measured with and without
addition of RT*
E
E
D
N/A
N/A
N/A
E
YES
E
E
E
E
D
D
N/A
N/A
N/A
N/A
N/A
N/A
Investigators’ lab
Materials and Methods
DNA
Materials and Methods
Manufacturer of reagents used and catalogue
number
Reaction volume (for two step reverse
transcription reaction)
Storage of cDNA: temperature, concentration,
duration, buffer
dPCR TARGET INFORMATION
Sequence accession number
D
N/A
D
N/A
D
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 and
GC content
Location of each primer by exon or intron (if
applicable)
Where appropriate, which splice variants are
targeted?
dPCR OLIGONUCLEOTIDES
Primer sequences and/or amplicon context
sequence**
RTPrimerDB Identification Number
Probe sequences**
Location and identity of any modifications
Manufacturer of oligonucleotides
Purification method
dPCR PROTOCOL
Complete reaction conditions
Reaction volume and amount of
RNA/cDNA/DNA
Primer, (probe), Mg++ and dNTP
concentrations
Polymerase identity and concentration
D
E
E
D
YES
YES
YES
YES
D
D
YES
N/A
Available on request
E
YES
Additional file 6
E
N/A
E
YES
Additional file 6
D
D
E
D
D
N/A
YES
YES
YES
YES
Additional file 6
Additional file 6
Materials and Methods
HPLC
E
E
YES
YES
Materials and Methods
Materials and Methods
E
YES
Materials and Methods;
Manufacturer’s proprietary
E
YES
AmpliTaq Gold® DNA
Polymerase; concentration is
Manufacturer’s proprietary
Buffer/kit Catalogue No and manufacturer
Exact chemical constitution of the buffer
Additives (SYBR Green I, DMSO, etc.)
Plates/tubes Catalogue No and manufacturer
Complete thermocycling parameters
Reaction setup
Gravimetric or volumetric dilutions
(manual/robotic)
Total PCR reaction volume prepared
Partition number
Individual partition volume
Total volume of the partitions measured
(effective reaction size)
E
D
E
D
E
D
D
YES
NO
N/A
YES
YES
YES
YES
Materials and Methods
Manufacturers’ proprietary
D
E
E
E
YES
YES
YES
YES
Materials and Methods
Materials and Methods
0.85 nL
770 partitions (digital
Methylight)
3080 partitions
(MDRE/MSRE dPCR)
CDKN2A (p14ARF):
NC_000009.12
COL2A1: NC_000012.12
Additional file 6
Additional file 6
Available on request
None detected by BLASTn
Materials and Methods
Materials and Methods
Materials and Methods
Manual
Partition volume variance/standard deviation
Comprehensive details and appropriate use of
controls
Manufacturer of dPCR instrument
dPCR VALIDATION
Optimisation data for the assay
Specificity (when measuring rare mutations,
pathogen sequences etc.)
Limit of detection of calibration control
If multiplexing, comparison with singleplex
assays
DATA ANALYSIS
Average copies per partition (λ or equivalent )
dPCR analysis program (source, version)
Outlier identification and disposition
Results of NTCs
D
E
N/A
YES
E
YES
D
E
YES
N/A
D
E
N/A
YES
E
E
E
E
YES
YES
N/A
YES
Examples of positive(s) and negative
experimental results as supplemental data
Where appropriate, justification of number and
choice of reference genes
E
YES
E
YES
Where appropriate, description of normalisation
method
Number and concordance of biological replicates
Number and stage (RT or qPCR) of technical
replicates
Repeatability (intra-assay variation)
E
YES
COL2A1 assay has been
previously described as an
effective methylation
independent reference
control gene for Methylight
[40,56,57].
Materials and Methods
D
E
N/A
YES
Materials and Methods
E
N/A
See below reproducibility
analysis
Reproducibility (inter-assay/user/lab etc.
variation )
Experimental variance or confidence interval***
Statistical methods used for analysis
D
YES
E
E
YES
YES
Standard deviation
measurements in Results
Results and Additional file 4
Data submission using RDML
D
N/A
Materials and Methods and
Results
Materials and Methods
Available on request
Results and Discussion,
Additional file 9
Results, Additional file 4
Materials and Methods
All NTCs gave negative
results as shown in
Additional file 9
Additional file 9
Materials and Methods and
Results
All essential information (E) must be submitted with the manuscript. Desirable information (D) should
be submitted if possible.
* Assessing the absence of DNA using a no RT assay (or where RT has been inactivated) is essential
when first extracting RNA. Once the sample has been validated as DNA-free, inclusion of a no-RT
control is desirable, but no longer essential.
** Disclosure of the primer and probe sequence is highly desirable and strongly encouraged. However,
since not all commercial pre-designed assay vendors provide this information when it is not available
assay context sequences must be submitted [58].
*** When single dPCR experiments are performed, the variation due to counting error alone should be
calculated from the binomial (or suitable equivalent) distribution.