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Hello there
I have an assignment at university for Parasitology.

I have received a review article titled "Genomics of reproduction in nematodes: prospects for parasite intervention?" by Nisbet, A.J. Cottee, P.A. & Gasser, R.B.

I have to present a 10 minute powerpoint on the "main points" that this article discusses. I understand that it is reviewing sex-specific molecules, technologies to identify these all in the hope of developing anthelminitcs and control strategies.

However, I am struggling to understand the concepts within the article such as the different technologies used and the differences between gender-specific and gender-enriched etc.

I was hoping that you could help me understand the key points that this article is conveying.

Regards
Question Date: 2010-09-02
Answer 1:

1. Gender-specific vs gender-enriched:
Gender-specific would be something that is found in only 1 gender, such as a Y-chromosome in human males.
Gender-enriched seems to refer to genes and things that may be expressed much more in one gender, as compared with the other gender.

I figured that out by searching the pdf of the article for 'gender specific' and then for 'gender enriched.'

2. Technologies used: see Table 1:
a. Northern blot
According to wikipedia, Northern blotting involves: extracting the RNA; using electrophoresis to separate it by size; blotting the electrophoresis gel onto a piece of something like paper to transfer the RNA bands to the paper; soaking the paper with a solution containing radioactive or fluorescent pieces of DNA or RNA with known sequences; and seeing which RNA bands on the paper bind to the radioactive DNA or RNA. You would know what the radioactive or flluorescent pieces of DNA or RNA were used for in the cell, so then you would know which RNAs in your sample did those things.

Wikipedia says Prof. Southern did 'blots' with samples of DNA, and they were named Southern Blots. When people used RNA samples instead, they named them Northern Blots.

b. cDNA microarray
A microarray is a grid of samples in a 'plate' or on a slide. For example, there are 96-well plates that are rectangular and have a 9 x 12 grid of little sample wells. cDNA is DNA made in a test tube that is complementary to the RNA in the sample, so it's a way of studying the RNA without having the problems of the RNA getting degraded by enzymes on our fingertips.

Check out wikipedia on 'DNA microarray'. They talk about microarray grids on a microscope slide, with 40,000 spots on each slide. Fluorescent pieces of DNA with known sequences [the 'probes'] are arrayed on the slide, and then you add your cDNA samples [targets] to the slide and see how bright each of the 40,000 spots becomes. There is usually something that makes the probe spots become bright when they bind to the DNA or RNA targets in the sample, but I'll not get into that.

c. cDNA microarray / macroarray
Google says macroarrays are basically the same as microarrays but bigger; the paper doesn't say anything about macroarrays outside of Table 1. The 96-well plates are probably macroarrays these days.

d. RT-PCR [= reverse transcriptase PCR]PCR [the Polymerase Chain Reaction] is a nice way of finding a single DNA sequence in a sample, even if there're only 1 or a few pieces of DNA with that sequence. PCR makes bunches of copies of DNA with the sequence you're looking for, if that sequence is present in the sample. Kary Mullis got the Nobel Prize for figuring out how to do it; he was my Teaching Assistant when I was a grad student in Biochemistry at Berkeley.

But your sample is RNA, not DNA, so first you have to make cDNA copies of the RNA, using reverse transcrptase [RT]; and then you can do PCR on the cDNA to get lots of copies of DNA with the same sequence as your RNA [or the complementary sequence, which has the same info.]

e. real time RT-PCR
This is a new type of PCR, where you can watch the PCR reaction in real time. Originally we just ran the PCR reaction until we thought it had gone on long enough, and then we checked what was in the test tubes. Now people can watch how much DNA is in the test tube during the whole time that the reaction is going on. In PCR, [the Polymerase Chain Reaction], the polymerase enzyme doubles the amount of DNA with each reaction cycle, so it's called a chain reaction. Wikipedia for 'real time PCR' says you can compare the amounts of an RNA sequence in different samples with real time PCR. And the 'RT' part of real time RT-PCR is needed to turn your RNA sample into a DNA sample [a cDNA sample, to be more precise], as described above. PCR needs lots less RNA to get the info you want, as compared with Northern Blots.

f. microarray / real time RT-PCR
I can only guess what combo of techniques this involves. One would need to read Ref. 12 to find out. But it must be a combination of the techniques above. Google doesn't give any hits for it.

3. What's the paper about?
The paper is a review article, so they're writing about work that has already been published. They're saying that research on sex differences in these worms might lead to new better drugs for fighting our problems with the worms. This makes sense. They're comparing different technical approaches. They're also talking about the sex differences that were found through the research in the different papers. C. elegans is a 'famous' worm, as you probably know, so they're comparing the results with other worms to the results with C. elegans. When I was a grad student at UCSB, we visited the lab of the professor who was discovering each different cell in C. elegans and what its function was.

Good luck with your talk. It's hard reading sc


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