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 |