Band Quantitation - Bio-Rad's Quantity One (Jun/30/2011 )

Hello, all. I've been generating a ton of data recently, and it's time for me to begin quantitating it. Specifically, I need to quantitate bands on some formaldehyde gels and Northern blots. I was wondering if anybody else uses Quantity One from Bio-Rad. How do you define bands? Do you 'Add Bands' manually, or have it automatically Detect Bands? I can identify bands and extract the intensity profiles just fine, but I was wondering which methods you might use in the quantitation. My PI told me to manually adjust the brackets according to the intensity profiles, but I keep thinking in the back of my mind that this technique is discretionary!

I was fiddling around with the program today, and I found that by placing the markers for "Add Bands" less than 1 mm apart on the image, my data is radically (!!!) altered. How can one define and quantitate bands without arbitrarily adjusting the brackets manually? Barring that, any tips on successfully finding the Trace values for gels/blots etc?

Thank you all for your input!

You can define boxes around the bands (make the box as big as the biggest band you have) using the volume rectangle tool. Then control click the box to make a new one, place this over another band, repeat for as many bands as you want to quantify. Finally make one more box and put it somewhere that you can define as background. If you double click this box (might be shift-click or alt-click on PC), you should get a window appearing that has some options... check the box that says background, then OK. YOu can also set standards if you have any using this box.

Now go to the volume analysis tool and use that to give you the volume, the adjusted volume (box minus area of background) of the box, the density and ony other options you want. Make sure the background subtraction is set to local!

Hit OK.

You now have the some (hopefully) useable results.