Difference between revisions of "QLanners Week 12"

Revision as of 21:52, 18 November 2017

Definitions of 10 Terms

1. annealed
2. hybridization

Outline

Background Information

• Saccharomyces cerevisiae used as model organism for research
• Haploid Strain FY73 used in study
• Studies have been done at growth temperature of 37◦ C and 39◦ C.
• Upregulation of five genes in cold shock from 30◦ C to 10◦ C have been discovered.
• This study looked at more sever heat and hold shock.

Methods

• Four fractions of cells grown at 30◦ C
• The fractions each experienced a different change in temperature
  • Fraction 1: 4◦ C for 180 minutes
  • Fraction 2: 45◦ C for 15 minutes
  • Fraction 3: 37◦ C for 30 minutes
  • Fraction 4: Half at 37◦ C and other half at 45◦ C for 15 minutes
• After specified time interval, each fraction harvested and frozen in liquid nitrogen
• RNA extracted through the use of a Micro-Dismembrator, mixing with TRI- ZOL Reagent, and the method of Chomczynski and Sacchi
• Probe generation was done through the following steps
  • 60 μg total RNA from each fraction was annealed to oligonucleotide dT15
    • This was used as a template to radiolabel first strand
  • First strand cDNA radio labeled with 50 μCi of [α-33P]-dCTP using SuperScript II (reaction at 43◦ C for one hour)
  • RNA hydrolysed with NaOH (at 65◦ C for 30 minutes)
  • Probe purified with isopropanol
• Filters prehybridized in hybridization mix (5× SSC, 5× Denhardt’s solution and 0.5% SDS)
• Probe denatured at 100◦ C for five minutes
• Probe hybridized with arrays overnight (65◦ C)
• Washed two times (5 minutes and 20 minutes)
• Filter regeneration
  • Poured a boiling solution of 5 mM sodium phosphate (pH 7.5) and 0.1% SDS over the filters
• Filters exposed to storage phosphor screen for 24 hours
• Data from filters collected using a Phos- phorImager Scanning Instrument 425
• Data images analyzed using Array Vision software
• For each tested temperature environment, data from 4 hybridizations were analysed, using two different arrays and two different RNA samples
  • Two replica-spots per gene on each array
  • Total of eight replica-spots per gene were analyzed
• Statistical analysis carried out using MATLAB
  • Significance of variance classified as:
    • High significance (met the min-max separation criteria)
    • Medium significance (met the standard deviation separation criteria)
    • Low (the rest of the data)

Flowchart

Flowchart of Experimental Methods outlined in journal article

Results

• Figure 1: Biplot obtained using a bipole algorithm called correspondence analysis. The different lines indicate the different fraction environments. The black dots all indicate intensity signals for individual genes. The closer a gene is to a particular colored line, the more correlated that gene's expression is with that line's condition. Furthermore, the positive and negative values on the axis indicate whether the expression of the gene was up- or down-regulated.
• Table 1: Count of the number of genes whose expression level changed in each condition tested. Only genes whose expression changed to a high or medium statistical significance were counted.

Findings from Results

• Effects of temperature shock include both activation and repression on genes
• More downregulated genes than upregulated genes. This finding is consistent with previous research from Gasch et al. (2000).
• More gene's are associated with the heat shock than the cold shock
  • And the number of genes affected is related to the intensity of the heat shock (higher the temperature, the more genes affected)
• The pre-adaptation of a lower temperature of heat shock results in less upregulated genes. However, this is not the same for downregulated genes.
  • Suggesting different regulatory circuits control the upregulated vs downregulated genes