Table 2
Comparison of some commonly used methods for RNA detection
| Method | Protocol | Advantages | Disadvantages |
|---|---|---|---|
| Northern blot | ▸ Separate RNA on gel | ▸ Reliable | ▸ Usually involves radioactivity |
| ▸ Transfer (blot) to filter | ▸ Quantitative | ▸ Requires large amount of RNA | |
| ▸ Hybridise to probe | ▸ Reusable (limited number of times) | ▸ Sensitive to degradation | |
| Autoradiography or phosphoimaging to detect probe signal | |||
| RPA (RNase protection assay) | ▸ Hybridise radiolabelled RNA probe with RNA | ▸ Highly sensitive | ▸ Labour intensive |
| ▸ Digest un-hybridised probe with ribonuclease (RNase) | ▸ Quantitative | ▸ Non-reusable | |
| ▸ Separate protected probe on gel | ▸ Reliably distinguish similar sequences and splice variants | ||
| ▸ Detect signal by autoradiography or phosphoimaging | |||
| Polymerase chain reaction (PCR) methods | ▸ Make cDNA of mRNA by reverse transcription (RT) | ▸ Highly sensitive | ▸ Largely non-quantitative (with |
| ▸ Requires minimal RNA input | exception of competitive PCR | ||
| ▸ Amplify specific cDNA target by PCR cycling | intensive construction and prior | ||
| ▸ Visualise PCR products on gel and by Southern blot, if necessary | quantification of a competitor target molecule) | ||
| Real time PCR | ▸ Convert RNA to cDNA by RT | ▸ Highly sensitive | ▸ Requires expensive real time |
| ▸ Amplify specific cDNA target by PCR | ▸ Quantitative | fluorescence detection | |
| ▸ Detect product using internal fluorescent probe (e.g. TaqMan) or by fluorescent dye (e.g. SYBR | ▸ Product is monitored each PCR cycle (hence “real time”) | hardware | |
| green) | ▸ Requires minimal RNA input | ||
| Gridded array filters | ▸ DNA copies of specific genes spotted in | ▸ Allows analysis of several hundred | ▸ Requires large input of RNA |
| gridded array on filters | genes simultaneously | ▸ Relatively insensitive | |
| ▸ Probe array with labelled cDNA made by RT of | ▸ Many commercial companies offer | ▸ Semiquantitative (individual | |
| RNA from a particular source | pre-gridded filters (e.g. cytokine array) | results require verification) | |
| ▸ Visualise hybridised spots (signal is proportional to abundance of RNA corresponding to each spotted gene) | ▸ Can custom make arrays | ||
| Microarrays, (cDNA arrays, Gene Chip) | ▸ DNA copies of genes (or oligonucleotides) are spotted onto high density grid on glass slide | ▸ Allows simultaneous analysis of thousands of genes | ▸ Requires expensive hardware/software or commercial service costs |
| ▸ Hybridisation with labelled cDNA prepared by RT of source RNA. (Typically, dual hybridisation protocols with fluorescent probes are used to look for global differences between two or more RNA sources) |
