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 |