RT-analyzer

An app to analyze real-time PCR data

Learn how to use RT-Analyzer on our Wiki
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Background

qRT-PCR or quantitative real-time PCR is a technique that is used to analyze expression level of a specific RNA molecule in a sample.

We won't go into too much detail here, but a PCR (polymerase chain reaction) is performed and the amount of DNA is measured at every cycle using fluorescent dyes. As the amount of DNA doubles after each PCR cycle, the fluorescence signal increases proportionally. At the end of the reaction, a threshold signal value is set and the cycle number at which the reaction passed the threshold value is calculated (CT). If the reaction started with more copies of the target molecule, the threshold will be reached earlier; if it started with less, the threshold will be reached later. Difference of 1 cycle corresponds to 2-fold difference between the target molecules in the beginning mixes.

If you would like read more about qRT-PCR, we recommend this paper from Nature Protocols.

Multiple reactions are run in parallel in order to test multiple samples (e.g. control vs treatment) and different genes in those samples. The difference between cycle numbers where a reaction passes a set threshold DNA amount, shows the difference between expression levels of two genes.

Why are we building RT-Analyzer?

There is no widely used, specialized program for RT-PCR analysis. People usually use Excel, which means a lot of time moving numbers around, typing in formulas etc. When the reaction numbers increase the sheets become hard to control. Finally, drawing the graphs in Excel can be a hassle. Adding error bars is not a simple task.

RT-Analyzer is a free, open-source and platform-independent web app that allows researchers perform qRT-PCR analysis in their browsers in seconds.

Input

Every reaction has four parameters: Location (coordinates of the reaction in a plate; A1, B7, C10 etc.), CT (the number of PCR cycle where the reaction passes a threshold DNA amount as measured by the fluorescent signal), Sample and Gene.

Different instruments have different output models, but a user can easily copy and paste their data into a .csv file in a predetermined format ("Location","CT","Sample","Gene"). RT-Analyzer accepts .csv files as input.

In addition to the data, the user can define an "internal control gene", "biological replicate groups" and a "control sample" or "control group".

Calculations

Relative expression level of a Gene is determined in relation to the internal control gene selected by the user using the deltaCt method described in the above mentioned paper.

Output

RT-Analyzer currently returns a list of averages and stdevs for each "Sample"-"Gene" pair in a table that can be copied and transferred into an Excel file.

Alternatively, RT-Analyzer can also draw graphs, which can be saved as an image. We are working on improving this feature.

Conclusion

RT-Analyzer is currently available as an alpha version. We would like people to give it a try and let us know how it worked out. Please send your feedback to rtanalyzer@gmail.com