MIQE checklist for authors, reviewers, and editors.1
Item to check | Importance | Item to check | Importance |
---|---|---|---|
Experimental design | qPCR oligonucleotides | ||
Definition of experimental and control groups | E | Primer sequences | E |
Number within each group | E | RTPrimerDB identification number | D |
Assay carried out by the core or investigator’s laboratory? | D | Probe sequences | D4 |
Acknowledgment of authors’ contributions | D | Location and identity of any modifications | E |
Sample | Manufacturer of oligonucleotides | D | |
Description | E | Purification method | D |
Volume/mass of sample processed | D | qPCR protocol | |
Microdissection or macrodissection | E | Complete reaction conditions | E |
Processing procedure | E | Reaction volume and amount of cDNA/DNA | E |
If frozen, how and how quickly? | E | Primer, (probe), Mg2+, and dNTP concentrations | E |
If fixed, with what and how quickly? | E | Polymerase identity and concentration | E |
Sample storage conditions and duration (especially for FFPE2 samples) | E | Buffer/kit identity and manufacturer | E |
Nucleic acid extraction | Exact chemical composition of the buffer | D | |
Procedure and/or instrumentation | E | Additives (SYBR Green I, DMSO, and so forth) | E |
Name of kit and details of any modifications | E | Manufacturer of plates/tubes and catalog number | D |
Source of additional reagents used | D | Complete thermocycling parameters | E |
Details of DNase or RNase treatment | E | Reaction setup (manual/robotic) | D |
Contamination assessment (DNA or RNA) | E | Manufacturer of qPCR instrument | E |
Nucleic acid quantification | E | qPCR validation | |
Instrument and method | E | Evidence of optimization (from gradients) | D |
Purity (A260/A280) | D | Specificity (gel, sequence, melt, or digest) | E |
Yield | D | For SYBR Green I, Cq of the NTC | E |
RNA integrity: method/instrument | E | Calibration curves with slope and y intercept | E |
RIN/RQI or Cq of 3′ and 5′ transcripts | E | PCR efficiency calculated from slope | E |
Electrophoresis traces | D | CIs for PCR efficiency or SE | D |
Inhibition testing (Cq dilutions, spike, or other) | E | r2 of calibration curve | E |
Reverse transcription | Linear dynamic range | E | |
Complete reaction conditions | E | Cq variation at LOD | E |
Amount of RNA and reaction volume | E | CIs throughout range | D |
Priming oligonucleotide (if using GSP) and concentration | E | Evidence for LOD | E |
Reverse transcriptase and concentration | E | If multiplex, efficiency and LOD of each assay | E |
Temperature and time | E | Data analysis | |
Manufacturer of reagents and catalogue numbers | D | qPCR analysis program (source, version) | E |
Cqs with and without reverse transcription | D3 | Method of Cq determination | E |
Storage conditions of cDNA | D | Outlier identification and disposition | E |
qPCR target information | Results for NTCs | E | |
Gene symbol | E | Justification of number and choice of reference genes | E |
Sequence accession number | E | Description of normalization method | E |
Location of amplicon | D | Number and concordance of biological replicates | D |
Amplicon length | E | Number and stage (reverse transcription or qPCR) of technical replicates | E |
In silico specificity screen (BLAST, and so on) | E | Repeatability (intraassay variation) | E |
Pseudogenes, retropseudogenes, or other homologs? | D | Reproducibility (interassay variation, CV) | D |
Sequence alignment | D | Power analysis | D |
Secondary structure analysis of amplicon | D | Statistical methods for results significance | E |
Location of each primer by exon or intron (if applicable) | E | Software (source, version) | E |
What splice variants are targeted? | E | Cq or raw data submission with RDML | D |