Introduction
DNA Sequencing
RNA Isolation & Detection
Acknowledgements

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 

 

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RNA Isolation and Detection Protocols for the Undergraduate Laboratory
Yeast mating as model for studying signaling pathways
There are a number of components that are important when designing a laboratory course. On the instructor's side these include accessibility for students, safety, theoretical concepts covered and techniques introduced and, unfortunately, amount of preparation time and cost. The students look for relevance, ease of understanding and timeliness (6hr labs are not favored).

Studying yeast mating can satisfy all of these criteria. Yeast grow quickly, and are relatively inexpensive to manipulate and maintain. Protocols exist, or can be adapted, so that cutting edge methods and theories can be explored and tested safely. And somehow studying mating (ie sex) in an organism responsible for alcohol production seems to pique the interest of undergraduate students.

Yeast mating is a good model for understanding extracellular and intracellular signaling events. The non-motile yeast must communicate and touch before they can mate. How? Is the signal unidirectional or bi directional (do one or both mating types make it)? Does the extracellular signaling require receptors that cement random acts of contact (the velco approach). Is the signal soluble? Exploring these questions requires a microscope, microcentrifuge and a solid logical approach (with a healthy dose of controls).

Once the mating reaction pathway has been initiated, cells must change their gene expression pathways so that contact can be established and maintained, the cell cycle can arrest and fusion between the mating cells can occur. Many components of the signaling pathways have been identified. Using expression of these genes as markers students can determine which pathways are effected in novelly isolated mating mutants. Exploring these questions requires protocols that follow.

For information on using yeast as the basis of undergraduate research in genomics see the Genome Consortium for Active Teaching (GCAT).
 
Problems isolating RNA from yeast (and one solution)
To sum it up yeast have a cell wall. It has evolved to help protect them from environmental changes and does a good job of keeping their RNA in and you out. When using a phenol-based isolation protocol this is not a problem. But, if 32 fledgling scientists pipetting phenol sounds like a bad idea to you there are a growing number of phenol-free RNA isolation kits available on the market. The problem is these kits are not optimized for use with yeast (some say they are but a call to tech support reveals otherwise) and if you do not breach the cell  wall  yield and quality of RNA decreases greatly.

We chose a combination of enzymatic activity (lyticase) and brute force (glass beads). In our hands neither one was enough alone but the combination works well.

Caveat: If you are studying other cellular processes you may need to use a different approach. Lyticase weakens/removes the cell wall without killing the cell. (In fact-given time the cells will repair their cell walls) It is very likely that the procedure induces stress response genes. When studying mating reaction genes this is not a problem--if your labs include examining stress response you'll have to change this step. A possible replacment is cycles of quick freeze/thaw (preferrably in liquid nitrogen but  dry ice or time in the -80C should work). We haven't tried this approach yet.
 
Phenol Free RNA isolation
To induce the mating response incubate mating type a cells in the presence or absence of alpha factor (synthetic peptide commercially available). Cells are pelleted by centrifugation and the cell wall removed by treatment with lyticase (15'x 30C). Lysis is accomplished cycles of vortexing/ice in the presence of acid washed glass beads. Cellular debris is removed by centrifugation and the supernatant is used as the sample for the Total RNA Safekit (Q-Biogene). Beginning with 30ml of log phase cells students routinely isolated 200ug of total RNA. Entire isolation/ quantification and blotting procedure can be completed in 2hr and 40min.
 
You got it--how good is it?
RNA quantity and quality can be determined by spectrophotometry and agarose gel electrophoresis (quick gel).

Spectrophorotmetry:
Using disposable UV/vis cuvettes (Fisher) determine the OD260 and OD280 of sample. The disposable cuvettes can be washed and reused. They aren't as good as quartz but they are very good and much less expensive.

Purity: Ratio of 280/260 between 0.4 to 0.55 indicates sample is 'clean' (has little protein contamination).

Sample Concentration: (OD260*dilution factor)/24 = ug/ul sample

Agarose Quick Gel:
This is a 'quick and dirty' way to check samples and see if DNA contamination is present.
1% agarose 1x TAE or 1xTBE gel using DNA markers, 'normal' (your favorite) DNA loading dye and EtBr staining. Run between 2 and 5ug of total RNA for approximately 20min at 120mV. Be careful: 10ug is not better--overloading will make it look as though sample degradation has occured.

Looking for ribosomal bands of approximately equal intensity and absence of low molecular weight band (degradation products). Genomic DNA may be detected as high MW band. gDNA is not a problem for Northern or dot blots but may require removal if sample is being used for RT-PCR (in genomic research)
 
What's it good for?

  • Traditional Northern blots
  • Dot or Slot Blots (faster version of a Northern--RNA is not separated by gel electrophoresis so no need for formaldehyde-formamide gels or the time they take)
  • RT-PCR
  • cDNA synthesis
Detecting specific RNA's --Under construction!!
If detecting RNA's by PCR based method, specific primers and PCR conditions will need to be optimized on a case-by-case basis.

We were interested in examining differential gene expression between a cells that had been exposed to alpha mating factor and a cells that were not undergoing the mating response. Dot blots were prepared using 10ug of total RNA. The nylon membranes were UV crosslinked and allowed to air dry. Prehybridization and hybridization were performed using temperatures optimal for the probes of interest. These protocols are being optimized during the spring semester of 2001 and will appear in an update of this site.



 
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