Image J for PCR analysis - File type (Sep/01/2008 )

Hi,

I am just about to start analysing a bunch of gels from PCR and just wanted to check a couple of things before I do them all wrong!! I think I understand the actual process from what I have read on here and on the Image J website. The thing I'm not sure about is the format my files should be in before I analyse them.

I have been saving them as TIffs, is the correct?
Do I then just open them as a regular Tiff file and bash on with the analysis?
Or should I invert them or save them as grey scale images or something?

Everyone else in my lab still uses Scion but I would rather use Image J as it is still updated and things. Any help would be fab

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Yes tiff files are fine. Just open them directly with Image J. Then you can greyscale them with ImageJ.

The procedure I use is as follows (mostly copy-pasted from Image J helpsites, probably the same ones you looked on ). It works OK:

3 Open your file with Image J

4 Under Image>Type click on 8-bit to convert the image to grayscale.

5 Go to the menu Process>Subtract Background. Use a rolling ball radius of 50. This removes some of the background coloration from your image.

6 Go to Analyze>Set Measurements, and click the boxes for Area, Mean Gray Value, and Integrated Density.

7 Go to Analyze>Set Scale, and enter "pixels" in the box next to Unit of length.
(If you have white bands and dark film you can Go to Edit>Invert (or hit Ctrl+Shift+I) to invert the colors.)

8 Choose the Rectangular Selection tool. Draw a rectangle around your first lane. Encompass some area of the lane above and below the band of interest.

9 Press the 1 button (or go to Analyze>Gels>Select First Lane). A new window will pop up with a copy of your image and a label over your first rectangular selection.

10 Use the arrow keys to move the rectangle over the next lane. Press 2 (or go to Analyze>Gels>Select Next Lane) to place a selection around the lane.

11 Repeat this for each lane on the membrane, moving the box and pressing 2 to place the selection.

12 When finished, press 3 (or go to Analyze>Gels>Plot Lanes), which pops up a new window with a profile plot of each lane.

13 Now choose the Straight Line selection tool. At the base of each peak, draw a line from one side of the peak to the other. This encloses the area of the peak. The tails to either side of the peak are the background signal. Note that if you have many lanes, the later lanes will be hidden at the bottom of the profile plot. To see these lanes, press and hold the space bar, and use the mouse to drag the profile plot upwards.

14 When each peak has been closed off at the base with the Straight Line tool, choose the Magic Wand (Wand tracing tool) from the tool palette.

15 Drag the profile plot back down until you are at the top peak. With the wand, click inside the peak. Repeat this for each peak as you go down the profile plot.

16 The results pop up in a result window. Copy Paste the results to Excel.

17 Save the graphs by choosing "Save as jpg. For the file name you MUST enter .jpg after the file name. (For some reason the program cant add the file extension).

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Hi,

Thanks so much for your advice. I am a bit confused about the background, if I subtract it before I invert the image then I see no bands at all! It seems to work OK if I invert it and then remove the background though, should I just do it in that order?

Helen

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hi,

if you just need to measure area of each of the peaks, do not need to substract the background before analysis...
(please refer to the 1st topic of this subforum "how to use ImageJ" for more info;)

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