TABLE 2.
Quality and application with MIQE guidelines. Modified from Nour et al. (2014).
| Items | Analysed Parameters | Method of Analysis |
|---|---|---|
| Assay details | Primer (probe) sequences/assay ID | “yes” if primer (and probe) sequences are provided |
| PCR efficiency | “yes” if this is an assessment of amplification efficiency | |
| Assay specificity | “yes” if there is mention of in silico homology search (BLAST, ePCR, BiSearch, or alike) | |
| Reverse transcription | Input amount of RNA in RT reaction | “yes” if input amount of RNA in RT reaction is mentioned |
| RT enzyme or RT kit | “yes” if there is any mention of reverse transcriptase used or specific kit, along with minimal instructions (can be according to manufacturer) | |
| Priming method | “yes” if the type of primers is mentioned (random primers, oligo-dT, blend, gene-specific primers,…) | |
| PCR | PCR conditions | “yes” if PCR conditions are listed or referred to an older publication |
| Taq polymerase or PCR kit | “yes” if there is any mention of Taq polymerase used or specific kit, along with minimal instructions (can be according to manufacturer) | |
| Final primer concentration | “yes” if final primer concentration in the reaction is mentioned (or can be deduced) | |
| Input amount template in PCR reaction | “yes” if input amount of template is mentioned | |
| qPCR validation | Evidence of optimization | ‘yes’ if protocol optimization outlined |
| Evidence of LOD (analytical sensitivity) | “yes” if the mention of serial dilution of known target |