Editing BioMicroCenter:RTPCR Protocol for Sample QC (section)

== Result Analysis ==

When run is complete use standard Cp (cycle number at which the amplification curve crosses the threshold) analysis for the machine being used.  

<span style="color: red">If using LichtCycler 480 remember to change “High Confidence” to “High Sensitivity” in results page, this is to allow for a more stringent threshold for quantifying Illumina libraries.</span>

Look over the amplification plots.  If successful amplification has occurred you should see typical sinusoidal amplification for each plot.  If the curves start below the 0 mark on the y-axis and appears to amplify around cycle 6, the sample is most likely too concentrated for the machine to accurately quantify the libraries.
[[Image:BioMicro_Amp_graphs.jpg|700px]]

===Calculate the Standard Curve:===
* graph log concentration vs. Cp of your standards:

[[Image:BioMicroCenter_RTPCR_std1.jpg|400px]]

{|[[Image:BioMicro_Too_conc_ampl_plot.jpg|400px]]||[[Image:BioMicro_Standard_curve.jpg|400px|]]|}

* The r^2 for the standard curve must be >0.98 or the RT-PCR  should be repeated.  Omit outliers as necessary. 
<span style="color: purple">If using standards prepared on a previous day compare the trend line equation to ensure that error has not been introduced</span><BR><BR>

===Calculate the Concentration of the Unknown Samples:===

* Use the standard curve equation to calculate the log concentration of your unknown samples (x = log concentration, y = Cp determined by RT-PCR)

* Solve the log to calculate the concentrations of the unknowns.   Multiply the values calculated by your dilution factor (typically 1000) to obtain the concentration of your sample.