Methods:

Total RNA Isolation from Yeast

• This protocol was used to isolate enough total RNA from yeast to label for the microarray experiments.  To isolate the RNA, 5 steps were taken: 1.  Growing the Yeast Cultures, 2. Preparing Yeast Spheroplasts, 3. Preparing Yeast RNA, 4. Determining the concentration of yeast RNA, 5. Checking the RNA for degradation using gel electrophoresis.
•  Use RNAZap to get rid of RNases.
•  Used wildtype sample #2 and ZMS1Δ sample #1.
•  A Potassium Phosphate Sorbitol buffer, Beta-mercaptoethanol, and lyticase were used while making spheroplasts
•  RNAsafe kit from Qbiogene, along with isopropanol were used to isolate the RNA.
•  O.D. readings were taken to determine RNA concentration and 1.2% agarose gel was run 100V for 30 minutes to check for degradation.
•  Note: Step 1, instead of using 30mL, used as much as possible of yeast culture.  Step 16: used 750µL of wildtype and 700 µL of ZMS1Δ. Step 21: used 100 µL instead 200 µL of solution 3. Step 22: used 20 µL of solution 4. Under checking RNA for degradation, 2µg of RNA was used instead of 10µg.

Labeling Procedure for Microarray Analysis

•  The RNA from the previous step was reverse transcribed using the Genisphere 3DNA Array 350 Detection Kit.
• The primer used was a poly TTTTT primer that has a special 3DNA capture sequence at the end of it.
•  Total RNA Cy3 and Cy5 reverse transcriptase primers and nuclease free water were vortexed together for each RNA sample. The tubes were heated to 80°C and snapped cooled on ice.
•  Reaction mix was made with RT buffer, dNTPs, superase-In RNase inhibitor and RT enzyme. This solution was added to RNA primer mix and incubated at 42°C for 2 hours.
•  ZMS1Δ sample 1 was labeled with cy3-green dye, and wildtype sample1 labeled with cy5-red dye.
•  10 µL of wildtype total RNA and 5.1 µL of ZMS1Δ total RNA was used.

 2-Step Hybridization of the DNA Chip Using the 3DNA Array 350 Protocol

•  The cDNA was hybridized to the complementary cDNA's attached to the slide.
•  The first hybridization was attaching the cDNA to the slide, vial 7 a 2X formamide based hybridization buffer was used because it was suitable for lower temperature hybridization. The second hybridization was loading the dyes so results can be visualized.
•  Note: for step 2 of Hybridization 1, 50 µL was needed of hybridization solution using 10 µL of concentrated cDNA and 13 µL of nuclease free water. For step 3, after the 75°C bath it was transferred into a 45°C bath. For hybridization 2, step 4, 2.4 µL of each dye was used.

How to Analyze GCAT Microarray Data Once You Have It

• After the scanning of the DNA chip (13697727) and viewing the resulting images in Scanalyze it was determined that results were insufficient for further analysis. This slide contained ΔZMS1(green) and wild-type (red).
• The data from slide 104 from Fall of 2004 was used instead. This slide examined ΔZMS1(red) and wild-type (green).

Using MAGIC Tool to Explore Data

•  After using Scanalyze to grid the microarray slide, MAGIC tool was used to analyze the ratios of expression for each gene.

Extra Projects

Project #2

Suggested protocol change for “Labeling Procedure for Microarray Analysis” Section II: Reverse Transcription, Step 6.

Alternate Reaction Mix:

4 ul LTI 5X first-strand buffer
1 ul dNTP mix (Vial 4)
2 ul 0.1 M DTT
1 ul RNasin (40 units)
1 ul LTI Superscript RT enzyme, 200 units

· Use of this different master reaction mix could allow for better reverse transcription of the RNA samples so that the dye would bind better in the anteceding steps.

Suggested protocol change for “2-Step Hybridization of the DNA Chip Using the 3DNA Array 350 Protocol” Section 2 Pre-hybridization, Step 1.
Alternate prehybridization solution:
50 ml of 2X SSC / 0.1% SDS / 0.1% BSA

· Using Bovine serum albumin (BSA), instead of sonicated salmon DNA, may block non-specific DNA binding better increasing the chances of the yeast cDNA binding.

Suggested protocol change for “2-Step Hybridization of the DNA Chip Using the 3DNA Array 350 Protocol” Section 3 Hybridization, Step 9.
Alternate hybridization technique:
Place in dry incubator at hybridization temp (45° C) for 3 days.

· By increasing the hybridization time, the chance of hybridization of the cDNA to the microarray slide may be increased.

An alternate procedure is also presented at the GCAT website by David Kushner at Dickinson College.

· This alternative procedure has worked well in the past according to the GCAT website and may work well for this experiment is following years.

Project #8:

1. Before performing this procedure remove all invalid and flagged expression ratios from the expression ratios.

2. Create a new workbook in Excel.

3. Type “1” in A2, “2” in A3 and “3” in A4.

4. Highlight A2-4 and drag the bottom right corner down until it reaches 7392 (the number of genes on the 2004 slide).

5. In cell B1 type “2004 genes.”

6. Download and open the gene list from GCAT entitled Chips  1 - 230 (File for Orientation around Chip; excel file) for the 2004 slide.

7. Copy cells D22-7413 which contain the gene names and paste them into column B of the new workbook starting at row 2.

8. In cell C1 type”2004 ratios.”

9. Open the file with the expression ratios for the 2004 slide being used.

10. Copy the expression ratios into column C of the new workbook starting at row 2.

11. Download and open the gene list from GCAT entitled Gene  Info File for Yeast (excel formate for orientation) for the 2007 slide.

12. In cell D1 type “2007 genes.”

13. Copy cells A2-6234 which contain the gene names and paste them into column D of the new workbook starting at row 2.

14. In cell E1 type”2007 ratios.”

15. Open the file with the expression ratios for the 2007 slide being used.

16. Copy the expression ratios into column E of the new workbook starting at row 2 .

17. Select columns A, B and C.

18. Open the menu “Data” then the option “Sort” and a popup window will open.

19. Select Sort by “2004 Genes,” click the ascending option and the My list has… “header row” option.

20. Select columns D and E.

21.  Open the menu “Data” then the option “Sort” and a popup window will open.

22. Select Sort by “2007 Genes,” click the ascending option and the My list has… “header row” option.

23. Highlight all of the data in columns D and E and using the Hand tool move the data around o that the genes listed in column D match those listed in column B. The insert cell function can be used to shift the data down so that the genes match, it is found under the “Insert” menu and “Cells…” option with the “Shift cells down” option selected in the popup window.

24. Once all of the genes in columns B and D match select all of the columns A through E.

25. Open the menu “Data” then the option “Sort” and a popup window will open.

26. Select Sort by “Column A,” click the ascending option and the My list has… “header row” option. The genes will now be in the same order for 2004 and 2007. 

27. Open a new workbook and copy columns A, B, C and E into it.

28. Save the new workbook as a “tab delimited text file” with the extension “.exp” and load the file into a MAGIC tool project.

Project #8 comparison of genes between 2004 and 2007 slide.

Figure A. Graph produced in Magic Tool comparing expression levels of YPL219W (Pho85p cyclin XIV) between slide 104 from 2004 and slide G5 from 2007. Three other relevant Pho85 genes were selected however results were not found for all grids. The genes not found were YER059W (Pho85 cyclin V), YGL134W (Pho85 cyclin VII), YGL215W (cyclin like protein that interacts with Pho85).