QLanners Week 12

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