Table S2: MIQE checklist.
ITEM TO CHECK
EXPERIMENTAL 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
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
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
IMPORTANCE CHECKLIST
E
E
D
D
YES
YES
YES
YES
E
D
E
E
E
E
N/A
N/A
N/A
N/A
N/A
N/A
E
N/A
E
E
D
E
E
E
YES
N/A
N/A
N/A
YES
YES
E
YES
D
D
E
YES
YES
N/A
COMMENTS/WHERE?
Materials and Methods
Materials and Methods
Investigator's lab
Author Information
Materials and Methods
Materials and Methods
Materials and Methods
Materials and Methods, Supplementary
Figure 1
Nanodrop (Thermal Scientific)
Materials and Methods
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 catalogue numbers
Cqs with and without RT
Storage conditions of cDNA
qPCR TARGET INFORMATION
If multiplex, efficiency and LOD of each assay.
E
D
E
N/A
N/A
N/A
E
E
E
E
E
D
D*
D
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
E
YES
Sequence accession number
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
D
E
E
D
D
D
E
E
YES
YES
YES
YES
YES
YES
YES
YES
E
D
D**
E
YES
N/A
YES
YES
Supplementary Figure 3
Materials and Methods and Supplementary
Table 1
Supplementary Table 1
Supplementary Table 1
Performed previously in Sanders et al., 2011
Performed previously in Sanders et al., 2011
Performed previously in Sanders et al., 2011
Performed previously in Sanders et al., 2011
Supplementary Table 1
Arabidopsis thaliana landsberg variant
Supplementary Table 1
Supplementary Table 1
Supplementary Table 1
Manufacturer of oligonucleotides
Purification method
qPCR PROTOCOL
Complete reaction conditions
Reaction volume and amount of cDNA/DNA
D
D
YES
YES
Primers (SIGMA) and probes (ABI)
HPLC
E
E
YES
YES
Primer, (probe), Mg++ and dNTP concentrations
E
YES
Polymerase identity and concentration
E
YES
Buffer/kit identity and manufacturer
E
YES
D
E
D
E
D
E
NO
N/A
YES
YES
YES
YES
Materials and Methods
Materials and Methods
Materials and Methods, Manufactures
proprietory
AmpliTaq Gold® DNA Polymerase, UP
(Ultra Pure)
TaqMan® Gene Expression Mastermix (PN
4369016)
Manufactures proprietory
Hydrolysis probe quantification only
ABI 96-well plates (PN 4306737)
Materials and Methods
Manual setup
Materials and Methods (ABI and Fluidigm)
D
E
E
E
E
D
E
E
E
D
E
YES
NO
N/A
YES
YES
N/A
YES
YES
YES
YES
YES
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)
For SYBR Green I, Cq of the NTC
Standard curves with slope and y-intercept
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
Supplementary Figure 3
Supplementary Figure 2
No SYBR Green assays used
Supplementary Figures 2 & 3
Supplementary Figure 3
Supplementary Figure 3
Supplementary Figure 3
Supplementary Figure 3
Supplementary Figure 3
Supplementary Figure 3
If multiplex, efficiency and LOD of each assay.
DATA ANALYSIS
qPCR analysis program (source, version)
Cq method determination
Outlier identification and disposition
E
YES
Supplementary Figure 3
E
E
E
YES
YES
YES
Results of NTCs
E
YES
Justification of number and choice of reference genes
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
E
D
E
E
D
D
E
E
D
N/A
YES
N/A
YES
YES
YES
No
YES
YES
N/A
Materials and Methods
Materials and Methods
No outliers identified
Materials and Methods, Supplementary
Figure 2
Standard curve quantification
Standard curve quantification
Materials and Methods
Results
Results
Not applicable
Materials and Methods/Supplemental
Materials and Methods/ Supplemental
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.
**: 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.