20.109(F07): Journal article discussion
Journal articles to be discussed
- The news story by Erika Check called "RNA interference: hitting the on switch" published in Nature 2007 vol. 448 pp. 855-8
- The original paper by Long-Cheng Li et. al. "Small dsRNAs induce transcriptional activation in human cells" published in PNAS 2006 vol. 103 pp. 17337-42.
Questions to guide your reading
1. Read the news story by Erika Check first, noting particularly the reporter's description of the scientists motivation and path to RNA activation. You will compare this description to how the scientific authors describe their path to RNAa in the introduction to the paper they wrote. Keep track of other reactions you have to the news story. Does it raise any questions for you? Is there anything surprising? Would you characterize the events as comedy or tragedy or neither?
2. Next skim the whole scientific article.
This means read the abstract once. Read the first and last sentence of the introduction. Read the subdivision headings of the Results section. Look at all the figures and their legends. Read the first and last paragraph of the Discussion.
3. Now it's time to really comb through the data.
We'll focus on the reported results, including the supplemental information. To help you organize the material, a few links and tables are given here.
Experimental matrix
| Treatments | Promoters examined | Cell types examined |
|---|---|---|
| dsRNA Aza-C (demethylase) IFN-alpha2a (nonspecific inducer) |
E-cadherin p21 VEGF |
PC3 and DU45 (human prostate cancer cell lines) Hela (human cervical carcinoma) MCF-7 (human breast cancer line) HEK293 (embryonic kidney line) LNCaP (human prostate cancer) J82 and T24 (bladder cancer) |
Experimental techniques
1. Western analysis: see this link for refresher info.
Data can be presented like this
where the treatment variation is shown above the gel lanes, the intensity of the protein band is shown inside the boxes (a cutout of the whole blot to just show the relevant bands). GAPDH is a "housekeeping gene" whose level is rarely affected by experimental perturbations. Why is it important to include data for this gene product? Thinking back to when you performed your Western analysis, did you included a similar control?
2. RT-PCR (also sometimes called q-PCR): see this link for some description of RNA measurement techniques including RT-PCR. You might also want to learn a little about standard PCR if you're not already familiar with this technique. Most biology textbooks describe the technique or you could look at some animations of the process, for example here, just follow the links through “techniques” and "amplifying DNA”.
Data can be presented like this
For next time
- Calculate the volume for 4 ug, 2 ug, 1 ug and 0.5 ug of each RNA sample that you prepared. Show your work. Save a copy of your answer since you'll need to know this volume to perform your experiment.
- Please familiarize yourself with the basics of microarrays by reading NCBI's primer on the technique.
