Analysing Gene Expression and its Regulation

Just realised that I missed out this transcription lecture. Joy. At least we got a laugh at how the slides refer to the gene that you're studying as "Your Favourite Gene," and even gave it the acronym YFG, as though it was a scientific term that everyone should know.

Describe how mRNA is isolated from total RNA.

This is reasonably simple, given that mRNA has a poly-A tail, which other types of RNA do not. Hence, mRNA can be isolated by using a cellulose matrix attached to oligo dT chains. This is done in a chromatography column with 0.5M NaCl.

Describe the steps in Northern blotting, including probe synthesis.

Northern blotting is a technique used to identify transcripts (i.e. mRNA molecules). I've written about it before in a previous post about hybridisation techniques.

As also mentioned in that previous post, probes use colour change or radioactivity in order to be identified. Molecules such as digoxigenin can be attached to the base (in areas where it will not interfere with the base pairing). Antibodies with fluorescent tags can then bind to these areas. In radiation, the alpha phosphate (i.e. the phosphate attached directly to the 5' carbon) is radioactive. (It has to be this particular phosphate, as the beta and gamma phosphates are lost as pyrophosphate in the DNA synthesis reaction.) Radioactive probes can then be identified via autoradiography.

Describe the type of quantification that can be obtained from Northern blots.

Quantification obtained from Northern blots is relative quantification. I presume this means that it isn't used to determine exactly how many mRNA molecules there are, but rather how many there are in comparison to other samples. Essentially, the denser the labelled bands on the nitrocellulose membrane, the more target mRNA there is. The density of the bands can then be compared to determine which samples have more or less of the target mRNA.

Describe the steps in in situ hybridisation, microarrays and RT-qPCR and the information obtained from each of these technologies

In situ hybridisation, as mentioned in my previous post about hybridisation techniques, determines where transcripts are found within tissues or cells. To prepare the slides, tissues are chemically preserved and embedded in wax, the tissue is sliced thinly and attached to slides, and the wax is removed. The other steps (probe generation, pre-hybridisation, hybridisation, washes and detection) are similar to that of Northern blots.

Microarrays are a method used to analyse the expression of thousands of genes simultaneously. They can also be used to compare the expression of genes in two different populations. Step number 1 is to isolate the mRNA, while step number 2 is to convert the mRNA into cDNA using reverse transcriptase. During this process, nucleotides are labelled with a fluorescent dye. Different dyes are used for the different populations. cDNA molecules are then hybridised to probes on the chip. (Different spots on the chip represent different genes- these are determined beforehand.) The colour and intensity of fluorescence at each spot on the chip gives relative quantification of gene expression in the two populations. Microarrays can also be used to perform cluster analysis, which is identifying genes that show similar expression patterns under similar conditions.

RT-qPCR, or reverse transcription quantitative PCR, continues on from our good friend PCR (see this earlier post for more details). This works pretty much like normal RT-PCR, except fluorescent tags are used to synthesise the new cDNA molecules. The intensity of the fluorescence increases as the number of cycles increases due to the production of more and more products containing fluorescent tags. This fluorescence can be quantified (presumably with some kind of machine that can detect the fluorescence) and a "threshold" intensity level is used for comparing different samples. RT-qPCR is also done with standards of a known concentration in order to create a standard curve. This standard curve can then be used to determine the original concentration of the mRNA of interest. (Let me know if that was confusing and I'll try and explain it better.)

Describe the function of pre-hybridisation and what factors affect stringency in hybridisation techniques such as Northern blotting, in situ hybridisation and microarrays.

The slides seem to take "pre-hybridisation" to mean "blocking" of the nitrocellulose membrane (or whatever medium you're using). "Blocking" basically involves adding proteins such as casein (found in milk) in order to cover the membrane and stop probes from binding to it (since it's better if the probe just binds to the mRNA of interest and not the membrane).

"Stringency" is a term that basically refers to how "strict" the probe is with regards to binding: will it bind only if there is an exact match, or is it a bit lenient? One factor that affects stringency is temperature: at higher temperatures (but below the melting temperature of the mRNA/probe complex), nothing will anneal except for exact matches. As you decrease the temperature, more and more probes will anneal. A second factor that affects stringency is salt concentration, as positive ions tends to stabilise the mRNA/probe complex (due to association with the negatively-charged phosphate backbone). The lower the salt concentration, the less likely the probe will bind unless it's a perfect match; the opposite is true for a high salt concentration.

So that's transcription over! w00t! Now for half a semester on proteins and enzyme kinetics... :P