Table 2

Comparison of some commonly used methods for RNA detection

MethodProtocolAdvantagesDisadvantages
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 inputexception of competitive PCR
▸ Amplify specific cDNA target by PCR cyclingintensive construction and prior
▸ Visualise PCR products on gel and by Southern blot, if necessaryquantification 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▸ Quantitativefluorescence 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 filtersgenes simultaneously▸ Relatively insensitive
▸ Probe array with labelled cDNA made by RT of▸ Many commercial companies offer▸ Semiquantitative (individual
RNA from a particular sourcepre-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)