Table 1.

MIQE checklist for authors, reviewers, and editors.1

Item to checkImportanceItem to checkImportance
Experimental designqPCR oligonucleotides
 Definition of experimental and control groupsE Primer sequencesE
 Number within each groupE RTPrimerDB identification numberD
 Assay carried out by the core or investigator’s laboratory?D Probe sequencesD4
 Acknowledgment of authors’ contributionsD Location and identity of any modificationsE
Sample Manufacturer of oligonucleotidesD
 DescriptionE Purification methodD
 Volume/mass of sample processedDqPCR protocol
 Microdissection or macrodissectionE Complete reaction conditionsE
 Processing procedureE Reaction volume and amount of cDNA/DNAE
 If frozen, how and how quickly?E Primer, (probe), Mg2+, and dNTP concentrationsE
 If fixed, with what and how quickly?E Polymerase identity and concentrationE
 Sample storage conditions and duration (especially for FFPE2 samples)E Buffer/kit identity and manufacturerE
Nucleic acid extraction Exact chemical composition of the bufferD
 Procedure and/or instrumentationE Additives (SYBR Green I, DMSO, and so forth)E
 Name of kit and details of any modificationsE Manufacturer of plates/tubes and catalog numberD
 Source of additional reagents usedD Complete thermocycling parametersE
 Details of DNase or RNase treatmentE Reaction setup (manual/robotic)D
 Contamination assessment (DNA or RNA)E Manufacturer of qPCR instrumentE
 Nucleic acid quantificationEqPCR validation
 Instrument and methodE Evidence of optimization (from gradients)D
 Purity (A260/A280)D Specificity (gel, sequence, melt, or digest)E
 YieldD For SYBR Green I, Cq of the NTCE
 RNA integrity: method/instrumentE Calibration curves with slope and y interceptE
 RIN/RQI or Cq of 3′ and 5′ transcriptsE PCR efficiency calculated from slopeE
 Electrophoresis tracesD CIs for PCR efficiency or SED
 Inhibition testing (Cq dilutions, spike, or other)Er2 of calibration curveE
Reverse transcription Linear dynamic rangeE
 Complete reaction conditionsE Cq variation at LODE
 Amount of RNA and reaction volumeE CIs throughout rangeD
 Priming oligonucleotide (if using GSP) and concentrationE Evidence for LODE
 Reverse transcriptase and concentrationE If multiplex, efficiency and LOD of each assayE
 Temperature and timeEData analysis
 Manufacturer of reagents and catalogue numbersD qPCR analysis program (source, version)E
 Cqs with and without reverse transcriptionD3 Method of Cq determinationE
 Storage conditions of cDNAD Outlier identification and dispositionE
qPCR target information Results for NTCsE
 Gene symbolE Justification of number and choice of reference genesE
 Sequence accession numberE Description of normalization methodE
 Location of ampliconD Number and concordance of biological replicatesD
 Amplicon lengthE Number and stage (reverse transcription or qPCR) of technical replicatesE
 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 alignmentD Power analysisD
 Secondary structure analysis of ampliconD Statistical methods for results significanceE
 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 RDMLD