In order to confirm the effectiveness and quality of the qPCR process that was performed, there are several actions and subsequent data that must be presented. This includes explaining the specific method of checking that the process functioned, such as using a gel, direct sequencing of the genetic material, showing a melt profile, or from digestion by restriction enzyme. If SYBR Green I was used, then the Cq of the control group with no template DNA must be given. Further essential data includes the calibration of the machine curves with the slope and y intercept noted, the efficiency of the PCR process as determined from the aforementioned slope, the correlation coefficients (r squared) for the calibration curves, the dynamic range of the linear curves, the Cq found at the lowest concentration where 95% of the results were still positive (LOD) along with the evidence for the LOD itself, and lastly if a multiplex is used, then the efficiency and LOD must be given for each assay done. The extra desired information includes evidence given that qPCR optimization occurred by the use of gradients, the confidence intervals to show efficiency of the qPCR, and the confidence intervals for the entire range tested. Data analysis The final section of the guidelines involves information on how the analysis of the qPCR data was done. The essential parts of that include the program and program version used for the analysis, the method for how the Cq was determined, figuring out the outlier points in the data and how they are used or excluded and why, what results were found for the controls with no template genetic material, an explanation for why the reference genes used were chosen and why the number of them was chosen, the method used to normalize the data, how many technical replicates were included, how repeatable was the data within the assays, what methods were used to determine significance of the results, and what software was used for this part of the qualitative analysis. It is also desired to include information on the number of biological replicates and whether they matched the results from the technical replicates, the reproducibility data for the concentration variants, data on the power analysis, and lastly for the researchers to submit the raw data in the RDML file format. References Further reading Molecular biology Laboratory techniques Polymerase chain reaction