Haynes:Roche480 Analysis

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Roche 480 Data Analysis

You loaded your reaction plate into the machine, the protocol is completed, and now you have a lot of data. This guide provides tips on how to convert Cp values (raw data) into meaningful data.

Sample Editor

First, use the Sample Editor (blue button) to tell the software what is in each well. Sample Editor is your friend. You will understand why when you carry out the Analysis phase.

RELATIVE QUANTIFICATION
Use this mode when your experiment includes a loading control (like GAPDH or ACTB), a control cDNA sample (like "untreated cells," "untransfected cells," "before," etc.), and an experimental cDNA sample (like "treated cells," "transfected cells," "after," etc.)

1. Click the Sample Editor button.
2. Select Relative Quantification near the top of the window.
3. Select every well that has cDNA template X (e.g. experimental) in it. In the Sample text field, type a meaningful, short label for that cDNA sample (e.g., Treated or U2OS_E001)
4. Repeat the above step for each unique cDNA sample, and for the no-template control (use the label "nTc"), if you included one.
5. Select every well that has experimental cDNA (e.g., treated, transfected, U2OS_E001, etc.). Select Unknown.
6. Select every well that has the control cDNA (e.g., untreated, untransfected, U2OS_C001, etc.). Select Positive control/ Calibrator.
7. Select every well that has a common Target Gene (e.g., PDX1, mCh, GAPDH, etc.) and type a short meaningful label for that gene under "Target". DO NOT EDIT THE SAMPLE FIELD.
8. Select every well that has the reference gene primers in it (e.g., GAPDH or ACTB). Select Reference.
9. Select every well that has all other gene primers in it (e.g., PDX1, mCH, etc.). Select Target.
10. Click the 'Save icon (looks like a 3.5 inch floppy disk...who uses those anymore?). SAVE EARLY, SAVE OFTEN.
11. Now you are ready for data analysis. Click the ANALYSIS button.
    

Tips on other Sample Editor modes to be added soon

Analysis

ADVANCED RELATIVE QUANTIFICATION
Use this mode when your experiment includes a loading control (like GAPDH or ACTB), a control cDNA sample (like "untreated cells," "untransfected cells," "before," etc.), and an experimental cDNA sample (like "treated cells," "transfected cells," "after," etc.)

1. Click the blue Analysis button to enter analysis mode.
2. Select Advanced Relative Quantification for All Samples. Select "High Sensitivity" and leave all other values as default. Click the checkmark button. Ignore any warnings.
3. Activate the Manual Pairing tab.
4. If pairs were auto-generated, click through each one (e.g., A1/A4) to see if the pairings make sense. If not click "Clear".
5. For a Dual color analysis (using UPL probes) use the radio buttons to set the Targets and the References to the right filters (e.g. 465-510 or 533-580).
6. To create pairing, click the plus icon at the bottom of the pairings list. With the new pairing selected, do the following:
   1. In the plate layout area for Targets, use CONTROL-click to select both the unknown and the corresponding calibrator (e.g. Treated-PDX1 and Untreated-PDX1).
   2. In the plate layout area for References, use CONTROL-click to select both the unknown and the corresponding calibrator's reference wells (e.g. Treated-GAPDH and Untreatd-GAPDH)
   3. Click Apply.
   4. Repeat this procedure for every Target gene.
7. Click the Calculate button.
8. Activate the Results tab, then activate the Bar Chart tab.
   Note: How to interpret the bar chart: Each pairing shows four bars. From left to right, the first blue bar = Control 2^(Cp_ref - Cp_gene1) = "Control delta Cp"; the red bar = Control delta Cp / Control delta Cp = 1.00; the next blue bar = Experimental 2^(Cp_ref - Cp_gene1) = "Experimental delta Cp"; the next red bar = Experimental delta Cp / Control delta Cp = normalized value
9. To export all Cp values into a txt file, right-click anywhere in the table of results and select export.

Tips

1. If outlier wells (super low or high Cp) are affecting the mean, you can eliminate them from the calculations:
   1. Activate the Target Name tab
   2. Select the desired gene name and filter set
   3. Click the Show Abs Quant button. The absolute quantification results will color-code the wells based on what is designated as a real amplification curve and what is not.
   4. In the Samples list, double-click on an outlier to get rid of it (it will become unchecked)
   5. Return to Relative Quantification mode (blue button at the bottom of the window) and click calculate again to re-run the calculation and to view results (in the Results tab).