Materials and Methods
Wild type yeast,
and yeast containing either Δzms1, Δzms2, or
Δzms1/Δzms2 mutants were grown.
Yeast was harvested when it had an optical density of 1.5 to 2.5, read
at 600 nm. The yeast cells were then
harvested and the cell walls of the yeast enzymatically degraded to form
Spheroplasts. RNA was then isolated
using the RNAsafe kit from Qbiogene. RNA
was quantified using the nanodrop to record absorbance, and run on a gel to
check for the 18S and 28S ribosomal bands.
Changes in
protocol:
Our particular
group used a wild type and ΔZMS2 mutant.
In order to make
the labeled probe, we used the Genisphere 3DNA Array 350 detection kit. This reverse transcribes the mRNA into cDNA
using a PolyT primer, with a special dDNA capture sequence at the end. Cy3 (green) dye was used with one yeast type,
and Cy5 (red) dye was used with the other.
Changes in
protocol:
The Cy3 (green)
capture sequence was used in the Δzms2 mutant yeast, while the Cy5 (red)
was used in the wild type yeast.
The cDNA was
hybridized using a multi-step procedure.
First, the microarray slides were pre-hybridized, then
the DNA was hybridized to the microarray slide, a process which takes 24
hours. The slide was then washed, and
the dye was hybridized to the slide, in a DARK room. Finally, the slides are washed a final time
and sent to Dr. Cambell for sequencing.
Different Statistical Analyses were then performed on the data that was received.
Table 1. Summary of array slides used for analysis comparing Δzms2 knockouts to WT yeast. Each slide contains two copies of the yeast genome.
| Slide Number | Dye for Δzms2 | Dye for WT |
| 725 | Cy3 (Green) | Cy5 (Red) |
| 728 | Cy5 (Red) | Cy3 (Green) |
| Introduction | Materials and Methods | Results | Discussion | Literature Cited |