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)