Image contrast adjustments for quantitated Northern blot pictures - (Mar/02/2008 )
I have a northern blot which was repeatedly probed for different transcripts, including a housekeeping gene as a loading control. All were quantified with an IP screen and ImageQuant style software.
Densiometric quantification shows that one is regulated, the other is not regulated, after adjusting for loading using the housekeeping gene. The regulation is definitely real, I've done loads of other experiments showing that.
I am making a figure, and my problem is that in the blot I want to publish, the unregulated one looks like it is regulated, due to differences (2x - 3x) in loading, after I adjust the contrast in Photoshop.
Unfortunately, the regulated gene is upregulated in the lanes that were also slightly overloaded - so they all look like they are regulated after I increase the contrast, although only one is. The picture is really misleading as the results from the image quantification are always quite clear that there is no regulation for the bottom gene. (OK it steadily decreases left to right but doesn't show the dramatic peak in the middle of the upper gene.)
Maybe I should adjust the contrast for each gene in proportion to the numerical values from the quant results? Of course, not individually adjusting each lane, but rather individually adjusting each of the three images separately.
I've attached a pic of the bands after contrast adjustment.
Top : regulated gene
Middle: housekeeping gene
Bottom: unregulated gene
Thanks for your advice!
I agree with you the image is misleading. Although you can normalize the band density and present the quantitative data the image is still problematic to reviewers. According to rules of many journals, you cannot adjust image such as contrast or levels locally, any adjustment has to apply to a whole image. I think the best solution is to rerun a Northern blot gel or use real-time PCR to obtain quantitative data.
Are you sure all the bands are specific, not 18s or 28s rRNA?
instead of the picture, why not show a table of normalized data?
agree - show a table or graph.
The same Northern blot was stripped and re-probed - so there is no one image with all three transcripts. I am not sure about the rules in this situation.
It was a lot of work to re-probe the same blot for 7 different transcripts so I don't want to repeat it! But i will if i must...