Sanders et al
Table S1 MIQE checklist for authors, reviewers and editors.
IMPORTANC
CHECKLIS
COMMENTS/
E
T
WHERE?
ITEM TO CHECK
EXPERIMENTAL DESIGN
Definition of experimental and
Materials and
E
YES
control groups
Methods
Materials and
Number within each group
E
YES
Methods
Assay carried out by core lab or
D
YES
D
NO
Investigators lab
investigator's lab?
Acknowledgement of authors'
Not required by
contributions
journal
SAMPLE
Description
E
N/A
D
N/A
E
N/A
E
N/A
If frozen - how and how quickly?
E
N/A
If fixed - with what, how quickly?
E
N/A
E
N/A
Volume/mass of sample processed
Microdissection or
macrodissection
Processing procedure
Sample storage conditions and
duration (especially for FFPE
samples)
NUCLEIC ACID EXTRACTION
~1~
Sanders et al
Materials and
Procedure and/or instrumentation
E
YES
Methods
Name of kit and details of any
Materials and
E
YES
modifications
Methods
Materials and
Source of additional reagents used
D
YES
Methods
Materials and
Details of DNase or RNase treatment
E
YES
Methods
DNase treatment –
Contamination assessment (DNA or
E
YES
RNA)
available on request
Materials and
Nucleic acid quantification
E
YES
Methods
Materials and
Instrument and method
E
YES
Methods
Purity (A260/A280)
D
YES
Available on request
Yield
D
YES
Available on request
E
YES
Materials and
RNA integrity method/instrument
Methods
RIN/RQI or Cq of 3' and 5'
E
YES
Available on request
D
YES
Available on request
E
YES
Figure 3
transcripts
Electrophoresis traces
Inhibition testing (Cq dilutions,
spike or other)
REVERSE TRANSCRIPTION
~2~
Sanders et al
Materials and
Complete reaction conditions
E
YES
Methods
Amount of RNA and reaction
Materials and
E
YES
volume
Methods
Priming oligonucleotide (if using
Materials and
E
YES
GSP) and concentration
Methods
Reverse transcriptase and
Materials and
E
YES
concentration
Methods
Materials and
Temperature and time
E
YES
Methods
Manufacturer:
Manufacturer of reagents and
D
YES
Materials and
catalogue numbers
Methods
Cqs with and without RT
D*
N/A
Storage conditions of cDNA
D
N/A
qPCR TARGET INFORMATION
Supplementary
Sequence accession number
E
YES
Information
Assay sequence:
Location of amplicon
D
YES
Supplementary
Information
Assay sequence:
Amplicon length
E
YES
Supplementary
Information
In silico specificity screen
E
~3~
YES
Available on request
Sanders et al
(BLAST, etc)
Pseudogenes, retropseudogenes or
None detected by
D
YES
other homologs?
BLAST
Sequence alignment
D
YES
D
YES
Secondary structure analysis of
Available on request
Supplementary
amplicon
Information
Assay sequence:
Location of each primer by exon or
E
YES
Supplementary
intron (if applicable)
Information
What splice variants are targeted?
E
N/A
qPCR OLIGONUCLEOTIDES
Supplementary
Primer sequences
E
YES
Information
RTPrimerDB Identification Number
D
N/A
D**
YES
Supplementary
Probe sequences
Information
Location and identity of any
E
N/A
modifications
Materials and
Manufacturer of oligonucleotides
D
YES
Methods
Purification method
D
YES
HPLC
qPCR PROTOCOL
Materials and
Complete reaction conditions
E
YES
Methods
~4~
Sanders et al
Reaction volume and amount of
Materials and
E
YES
cDNA/DNA
Methods
Materials and
Primer, (probe), Mg++ and dNTP
Methods;
E
YES
concentrations
Manufactures
proprietary
Polymerase identity and
Materials and
E
YES
concentration
Methods
Buffer/kit identity and
Materials and
E
YES
manufacturer
Methods
Exact chemical constitution of the
Manufactures
D
NO
buffer
proprietary
Additives (SYBR Green I, DMSO,
E
N/A
D
YES
etc.)
Manufacturer of plates/tubes and
Lo Bind tubes
catalog number
(Eppendorf)
Materials and
Complete thermocycling parameters
E
YES
Methods
Reaction setup (manual/robotic)
D
YES
Manufacturer of qPCR instrument
E
YES
Manual setup
Materials and
Methods
qPCR VALIDATION
Evidence of optimisation (from
D
YES
Available on request
E
NO
Not possible after
gradients)
Specificity (gel, sequence, melt, or
~5~
Sanders et al
digest)
For SYBR Green I, Cq of the NTC
digital PCR
E
N/A
E
N/A
Digital PCR
E
N/A
Digital PCR
D
N/A
Digital PCR
E
N/A
Digital PCR
E
YES
Standard curves with slope and yintercept
PCR efficiency calculated from
slope
Confidence interval for PCR
efficiency or standard error
R2 of standard curve
Results and
Linear dynamic range
Discussion
Cq variation at lower limit
E
N/A
Digital PCR
D
YES
Figure 3
E
YES
Figure 3
E
YES
Figure 3
Confidence intervals throughout
range
Evidence for limit of detection
If multiplex, efficiency and LOD of
each assay.
DATA ANALYSIS
qPCR analysis program (source,
Materials and
E
YES
version)
Cq method determination
Methods
E
N/A
E
YES
Outlier identification and
Materials and
disposition
Results of NTCs
Digital PCR
Methods
E
~6~
YES
Table 1
Sanders et al
Justification of number and choice of
E
N/A
E
N/A
D
N/A
E
YES
reference genes
Description of normalisation method
Number and concordance of
biological replicates
Number and stage (RT or qPCR) of
Materials and
technical replicates
Methods
Materials and
Repeatability (intra-assay variation)
E
YES
Methods
Reproducibility (inter-assay
Results and
D
YES
variation, %CV)
Power analysis
Discussion
D
Figure 3
No
Statistical methods for result
Materials and
E
YES
significance
Methods
Materials and
Software (source, version)
E
YES
Methods
Cq or raw data submission using
D
N/A
RDML
All essential information (E) must be submitted with the manuscript. Desirable information
(D) should be submitted if available. If using primers obtained from RTPrimerDB,
information on qPCR target, oligonucleotides, protocols and validation is available from that
source.
*: Assessing the absence of DNA using a no RT assay 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.
~7~
Sanders et al
**: Disclosure of the probe sequence is highly desirable and strongly encouraged. However,
since not all commercial pre-designed assay vendors provide this information, it cannot be an
essential requirement. Use of such assays is advised against.
~8~