Transcript analysis

Transcript analysis is the collection of methods used in the detection, quantification and mapping of RNA transcripts.

All transcript analysis requires that RNA is first isolated from cell extracts and purified. The presence of the enzyme RNase, which would digest the RNA as it was purified, is a problem in these procedures, and often the apparatus must first be treated with strong denaturants (e.g. Trizol) and RNase inhibitors to minimise its effect. The denaturants are also needed to degrade any proteins that may be bound to RNA in ribonucleoprotein complexes. The cells are homogenised in a Trizol reagent for 5 minutes, and then chloroform is added to separate the phases (this takes around 20 minutes). Finally, RNA is precipiated and then washed and solubilised to be ready for analysis.

When isolating messenger RNA (mRNA) from cell extracts it is common to first centrifuge the cell contents in order to isolate ribosomes. Ribosomes carry mRNA that is fully processed and spliced, while the nucleus, for example, contains many incompletely-spliced mRNAs. Ribosomal RNA (rRNA) comprises about 80% of total cellular RNA, while transfer RNA (tRNA) comprises 15% and mRNA only 5%. In spite of this, eukaryotic mRNA is easily isolated by virtue of its 3' poly-A tail which can hybridise to an oligo(dT) column in the lab.

Various analyses that can then be performed include:

1. Identifying the transcription start site

2. Quantifying the amount of transcript

3. Detection of transcripts in vivo

4. Measuring the half-life of mRNA

mRNA half-life is dependent on both the rate of transcription and the rate of degradation. To measure this, first inhibit RNA synthesis using actinomycin D, then measure mRNA levels, using radio-probing, at regular time intervals following synthesis inhibiton.