Table 3.

Criteria that are required (*) or should be considered in real-time quantitative PCR methodologies.

RNA A260/A280 ratios are reported and are above 1.8 to indicate sample purity, or are consistent across samples The integrity of RNA was assessed (common strategies include an RNA integrity number (RIN), an RNA quality indicator (RQI) or 28s:18s ratio) to ensure minimal RNA degradation or consistency across samples Reference genes (also referred to as housekeeping genes) were used to normalize the data* There is evidence that reference gene expression was not affected by treatment* It is ensured that the primer used is specific to the gene of interest (common strategies include the use of melting curves, gel electrophoresis or sequencing)* PCR arrays generally contain pre-validated primers that are gene-specific No reverse transcription (NRT) controls were included to test for genomic DNA contamination of RNA samples Technical replicates were performed The number of cycles required to detect a true signal should be below 35* PCR efficiencies calculated using the slope of the standard curve are reported