genetics - How is gene expression estimated?

I'm reading this fantastic article on estimating body time: Molecular-timetable methods for detection of body time and rhythm disorders from single-time-point genome-wide expression profiles and one of the things that is not very clear to me is how the researchers estimated which genes are expressed and which ones are not:

Total RNA was prepared by using Trizol reagent (GIBCO􏱾BRL). cDNA synthesis and cRNA labeling reactions were performed as described (5). Affymetrix high- density oligonucleotide arrays (Murine Genome Array U74A, Version 1.0, measuring 9,977 independent transcripts) were hybridized, stained, and washed according to the Technical Manual (Affymetrix). Affymetrix software was used to deter- mine the average difference (AD) between perfectly matched probes and single-base-pair-mismatched probes. The AD of each probe was then scaled globally so that the total AD of each microarray was equal. The resulting AD values reflect the abundance of a given mRNA relative to the total RNA popu- lation and were used in all subsequent analyses

I'm not sure if I'm reading this correctly - did the researchers look at all RNA available in the cells and calculated the levels of messenger RNA produced by expressed genes? if not, how can the level of expression of a gene be estimated?

Answer

The technique described here is called microarray. Your question has given me an opportunity to put forth one of my opinions about certain problems of gene expression studies.

Gene expression is a measure of the activity of any gene. If the gene performs its activity in the form of a protein, then its expression should be a measure of the protein. If a gene makes a non-coding RNA then its expression is a measure of the RNA concentration.

[You can omit the cases of post-translational modification because of signal transduction. They are highly expressed and transiently but frequently used. ]

Like your example there are many studies which use mRNA concentration as a proxy for protein activity. This proxy works in many cases because transcriptional gene regulation is more frequently used mechanism for imparting stable changes. But the best strategy would always be to measure the proteins also.

Apart from microarray there are several techniques to measure RNA concentration:

• RNA sequencing (high throughput)


• Real-Time PCR (medium throughput)


• Northern Blotting (Low throughput, semi-quantitative)

Many techniques exist for measuring proteins also:

• Mass spectrometry (high throughput)


• ELISA (Low throughput, quantitative)


• Western blotting (Low throughput, semi-quantitative)

Usually the "proxy method" is used because protein quantification is comparatively more difficult. Antibody based techniques like ELISA and Western blotting have problems of cross comparison of proteins because of the variability of the antibody binding efficiency.