Regulation of Gene Expression - Methods

Methods

In general, most experiments investigating differential expression used whole cell extracts of RNA, called steady-state levels, to determine which genes changed and by how much they did. These are, however, not informative of where the regulation has occurred and may actually mask conflicting regulatory processess (see post-transcriptional regulation), but it is still the most commonly analysed (QPCR and DNA microarray).

When studying gene expression, there are several methods to look at the various stages. In eukaryotes these include:

• The local chromatin environment of the region can be determined by ChIP-chip analysis by pulling down RNA Polymerase II, Histone 3 modifications, Trithorax-group protein, Polycomb-group protein, or any other DNA-binding element to which a good antibody is available.
• Epistatic interactions can be investigated by synthetic genetic array analysis
• Due to post-transcriptional regulation, transcription rates and total RNA levels differ significantly. To measure the transcription rates nuclear run-on assays can be done and newer high-throughput methods are being developed, using thiol labelling instead of radioactivity.
• Only 5% of the RNA polymerised in the nucleus actually exists, and not only introns, abortive products, and non-sense transcripts are degradated. Therefore, the differences in nuclear and cytoplasmic levels can be see by separating the two fractions by gentle lysis.
• Alternative splicing can be analysed with a splicing array or with a tiling array (see DNA microarray).
• All in vivo RNA is complexed as RNPs. The quantity of transcripts bound to specific protein can be also analysed by RIP-Chip. For example, DCP2 will give an indication of sequestered protein; ribosome-bound gives and indication of transcripts active in transcription (although it should be noted that a more dated method, called polysome fractionation, is still popular in some labs)
• Protein levels can be analysed by Mass spectrometry, which can be compared only to QPCR data, as microarray data is relative and not absolute.
• RNA and protein degradation rates are measured by means of transcription inhibitors (actinomycin D or α-amanitin) or translation inhibitors (Cycloheximide), respectively.