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Table 1 Checklist for authors' of MIQE précis at time of manuscript submission, detailing information about individual parameters associated with each step of the RT-qPCR workflow.
| Sample/Template | details |
|---|---|
| Source | If cancer, was biopsy screened for adjacent normal tissue? |
| Method of preservation | Liquid N2/RNAlater/formalin |
| Storage time (if appropriate) | If using samples >6 months old |
| Handling | fresh/frozen/formalin |
| Extraction method | TriZol/columns |
| RNA: DNA-free | Intron-spanning primers/no RT control |
| Concentration | Nanodrop/ribogreen/microfluidics |
| RNA: integrity | Microfluidics/3':5' assay |
| Inhibition-free | Method of testing |
| Assay optimisation/validation | |
| Accession number | RefSeq XX_1234567 |
| Amplicon details | exon location, amplicon size |
| Primer sequence | even if previously published |
| Probe sequence* | identify LNA or other substitutions |
| In silico | BLAST/Primer-BLAST/m-fold |
| empirical | primer concentration/annealing temperature |
| Priming conditions | oligo-dT/random/combination/target-specific |
| PCR efficiency | dilution curve |
| Linear dynamic range | spanning unknown targets |
| Limits of detection | LOD detection/accurate quantification |
| Intra-assay variation | copy numbers not Cq |
| RT/PCR | |
| Protocols | detailed description, concentrations, volumes |
| Reagents | supplier, Lot number |
| Duplicate RT | ΔCq |
| NTC | Cq & melt curves |
| NAC | ΔCq beginning:end of qPCR |
| Positive control | inter-run calibrators |
| Data analysis | |
| Specialist software | e.g., QBAsePlus |
| Statistical justification | e.g., biological replicates |
| Transparent, validated normalisation | e.g., GeNorm summary |
