Identifying Transcripts

Northern blots are similar to Southerns; however, the target molecule is RNA rather than DNA. More specifically, RNA samples are isolated, separated by gel electrophoresis, immobilized on a membrane, and probed with a DNA bait sequence. Bound probe is detected with autoradiography or some other enzyme-based detection method. Northern blots are qualitative (i.e., transcript is present or absent within a sample), not quantitative (i.e., more or less transcript present), unless a loading control33 is included in the experiment. Thus, northern blots are ideal for answering questions such as “Is the transcript of interest expressed in this cell/tissue/organism under this condition?”

A modified version of PCR, called RT-PCR, can also be used to detect specific transcripts within a mixed RNA sample. DNA Pol used in PCR can only amplify DNA templates. To overcome this technical limitation, in RT-PCR, RNA samples are converted to cDNA then PCR amplified and separated by gel electrophoresis for detection. If a band appears at the end of an RT-PCR protocol,34 then the RNA molecule of interest was present in the original sample. RT-PCR is qualitative, unless a more advanced detection method is used. Quantitative RT-PCR (qRT-PCR) involves detecting the abundance of PCR product at the end of each cycle of the reaction. Thus, it involves “real-time” detection of products. Because of this, qRT-PCR is also called real-time qRT-PCR.

Reporter fusions are a third method for measuring transcript abundance. The primary difference between reporter fusions and northern blot or RT-PCR is that transcript abundance is measured in vivo rather than in vitro. Reporter genes encode proteins that are easy to detect. Common examples include green fluorescent protein (GFP; gfp) and its derivatives from jellyfish, beta-galactosidase (LacZ; lacZ) or beta-glucuronidase (GUS, gus) from E. coli, and luciferase from bacteria or fireflies. Reporter fusions are constructed to assess gene transcription. Like switching appliances plugged into a single socket, genes can be exchanged downstream of a promoter of interest. For example, suppose you were interested in the expression of your favorite gene (yfg). You could create a fusion between yfg’s promoter and gfp using rDNA technologies. Cells carrying the construct would glow green whenever yfg was typically transcribed. That is, expression of the reporter mirrors that of the original gene.

 
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