A sample is considered positive if the Ct value is less than the number of cycles performed. However this does not mean that quantitation is possible in every case. Accurate quantitation is only possible within the reproducible range of the assay, i.e. higher or equal to the reproducible sensitivity. For plasmid controls this is usually between five and ten copies.
'Negative specimens' are those where no amplification has occurred and the control gene amplification is satisfactory (see below). A sample can also be considered negative if the lowest Ct is within the range of the NAC controls or if all of its Ct values are more than 4 cycles apart from the highest Ct of the maximal sensitivity control.
Arbitrary cut-off values for control gene levels may be set. For RT-PCR in our laboratory we do not report a test sequence as 'undetected' if the cABL
copy number is less than 104 copies per microlitre of cDNA or if the mean Ct is greater than 30. Specimens that fail to achieve the above criteria would be re-purified and concentrated prior to reverse transcription, as the most common reasons for such failures are DNA contamination, low RNA concentration or RNA degradation. Examination of RNA integrity can be performed using the Agilent 2100 Bioanalyser (Agilent Technologies) or gel electrophoresis. However RNA degradation is rare in fresh samples properly processed. If the sample was more than 48 hours old at the time of processing some RNA turnover/degradation is inevitable, clinical reports of negative findings should include comments regarding time to receipt of specimens.
Was this article helpful?