Replies

• Craig Rowell

  30 October 2007 | 10:57

  Hi Dinesh,

  This is certainly a very important set of questions and I look forward to all the answers. In my opinion (only having experience with questions A and C) I would say “semi-quantitative”. For western-blot, especially with film, you have a limited dynamic range to analyze and likely the results are a relative comparison. That is why you have to run all of your sample set together on the same gel for proper comparison. The answer for proteomics is similar. The quantitative aspects are strictly for initial analyatical study. More sophisticated mass-spec is certainly excellent for quantifying highly pure samples, but not the mixed “proteome” set. Not even the new DIGE (differential in gel electrophoresis) dyes will give you truly quantitative numbers.

  As much as we would like to look at “reall” numbers, isn’t that part of the of the art of science – being able to interpret our results in the face of partial answers to set up our next hypothesis.

• Bronwen Dekker

  01 November 2007 | 22:32

  While I cringe slightly when I hear, for example, Western blotting being referred to as “quantitative” (as there are many techniques where the signal-to-concentration correlation is better and where you expect much smaller error bars), I think that if you are using the “best” modern equipment, being paranoid about keeping your sample within the dynamic range of the assay, and using appropriate internal and external standards Western blotting has probably reached a stage where it could be termed quantitative.

  A point that emerges from the latter statement is that an assay which starts out as being “just” qualitative tends to become “more quantitative” with time as more optimisation etc is done. At what point do we decide that a technique is quantitative? Are only techniques where you can determine the absolute concentration of the analyte quantitative, for example?

• Mike Hemberger

  12 January 2008 | 11:42

  Dear Dinesh,
  I think sytems biology is at least a first step. Some quantitative immunoblotting techniques are available now to my knowledge (see Klingmüller at the cancer research institute in heidelberg). To obtain a truly dynamical behaviour of involved proteins the sample over time is randomized in gel loading order and “calibrators” are added form which the concentration is known. Additionally it would be possible to obtain absolute amounts if samples are loaded from which one can access the signal strength with increasing concentration. If the quantify the results not on a film but with fluorescnece agents there is at least a quite linear quantification possible.

  Regards,
  Mike

• Paul Brennan

  20 February 2008 | 16:52

  Dear Dinesh,

  Systems Biology is not the only area that require quantitative biology. In fact quantitative biology is probably more important for clinical diagnostics. Clinicians regularly require absolute values of proteins to inform treatment and diagnosis.

  In order to be used for clinical decision, assays must be standardised and validated, something often difficult in research. To answer more directly:
  a) western blotting – almost never used for clinical info – probably at best semi-quantitative
  b) fluorescence based techiques are used for cell counting by flow cytometry. “Imaging” is used to get counts of cells with molecular mutations (genome mutations for cancer)
  c) proteomics tools – still in development so not now but maybe in the future
  d) RNA-omics tools – well quantitative RT-PCR is used for clinical decisions – again to quantify the levels of cancer genes.

  Bear in mind technologies, validated, that are regularly used for diagnosis:
  ELISA, RIA, electrolytes, cell counting by microscopy, enzyme assays of various kinds.

  Perhaps if we want to use proper systems biology we should look at validated techniques?
  Best wishes,
  Paul

• User removed

  26 March 2008 | 14:28

  This content has been removed by the forum moderators.