Bio 580 Project

Objective:

The goal of this experiment was to determine the expression of two genes in mutant ZMS2 compared to wildtype Saccharomyces cerevisiae by using realtime PCR (RT-PCR). This information will then be compared to the expression data of the genes using a microarray as to validate the results of the microarray technique. The two genes of interest were ACT1 and ALD6. ACT1 is a housekeeping gene (Zhang et al., 2000; Nailis et al., 2006) that codes for beta-actin, which plays a role in cytoskeletal processes such as endocytosis (Botstein et al., 1997) and cytokinesis (Bi et al., 1998). ALD6 codes for Cytosolic aldehyde dehydrogenase which produces acetate from acetaldehyde in the glucose fermentation pathway (Eglinton et al., 2002 and Saint-Prix et al. 2004).

Methods:

RNA Isolation

The same isolated RNA used for the microarray experiment was used for this experiment. The procedure for this technique can be found here. 

cDNA Creation

Reverse transcription of the RNA into cDNA was done using a Genisphere 3DNA Array 350 Detection Kit. cDNA was created using the RNA isolated from mutated ZMS2 and wildtype Saccharomyces cerevisiae. Four samples were set up, two controls (one wildtype and one mutant) containing just RNA and two cDNA samples (one wildtype and one mutant). The controls were created by not adding reverse transcriptase to the RNA sample. Each sample was then concentrated using YM30 microconcentrators.  The protocol for reverse transcription and cDNA concentration can be found here. Changes to the protocol included not using 4ul of 0.1M DTT and 4ul of 50mM MgCl2, using 1ul of 10uM DNTP mix, and 1ul of RT enzyme.

Primers

Primers were developed for ACT1 and ALD6 so that a RT-PCR experiment of these cDNAs could be perforemed. Primer sequences for ACT1 and ALD6 were generated using the internet program Primer3. The program used the ACT1 and ALD6 gene sequences to produce proper forward and reverse primers. The primers were then created by Integrated DNA Technologies, Inc. (Coralville, IA). The sequence of the forward ACT1 primer is TGTCACCAACTGGGACGATA. The primer has a Tm of 55.7°C and a 50% GC content  The sequence of the reverse ACT1 primer is AACCAGCGTAAATTGGAACG. The primer has a Tm of 53.7°C and a 45% GC content. This primer set produces a product of 281bp. The sequence of the forward ALD6 primer is CAGAAAGCTGGCTTTTACCG. The primer has a Tm of 54.3°C and a 50% GC content. The sequence of the reverse ALD6 primer is AGCAGCCAATAGTTCGTCGT. The primer has a Tm of 56.7°C and 50% GC content. The product produced by this primer set is 250bp.

RT-PCR

RT-PCR was used to quantitate the cDNA of the four samples. Primers were diluted to a 10uM concentration with sterile water. A RT-PCR mastermix was created for each gene by adding 50ul of 2 x PCR supermix, 2ul of 10uM forward primer, 2ul of 10uM reverse primer , and 30.5ul of sterile water. 16.9ul of the mastermix with 3.1ul of cDNA, RNA, or water (control) was added to a RT-PCR tube. This was done for the mutant and wildtype samples. The following RT-PCR protocol was used:

• 94 C for 10 min.
• 94 C for 10 sec.
• 50 C for 20 sec.
• 72 C for 1 min. 20 sec.
• 76 C for 1 sec
• plate read
• 78 C for 1 sec
• plate read
• 80 C for 1 sec
• plate read
• 82 C for 1 sec
• plate read
• 84 C for 1 sec
• plate read
• 86 C for 1 sec
• plate read
• 72 C for 10 min
• 72 C for 10
• 4 C degrees forever

*adapted from T. Rife RT-PCR protocol
 

Results and Discussion:

        Results of the RT-PCR allowed quantitation of the cDNA concentrations in each sample. In order to quantitate only the genes of interest and not any other molecules (primer dimers) a melting curve was used to determine the Tm of each product (Figure 1).  The optimal melting temperature (Tm) for quantitating the cDNA in our samples was determined to be 80°C. At this point all the primer dimers would have melted but the desired cDNA would remain annealed. Exact Tm's were hard to distinguish because peaks for ALD6 samples were not distinct.

                                               ACT1                                                                           

                                               ALD6

Figure 1. RT-PCR melting curve derivative of ACT1 and ALD6 PCR samples. PCR samples were obtained from RNA extracted from mutated ZMS2 and wildtype Saccharomyces cerevisiae. Temperature can be read on the x-axis and quantum yield (fluorescence) is measured on the y-axis. Samples contained 3.1ul of RNA or cDNA and the control was a water blank.   

 Analyzing the PCR quantitation curves of the RT-PCR samples revealed Ct values for each sample (Figure 2). By doing this a comparison of the amount of double-stranded cDNA can be made between the samples. The amount of DNA is inversely related to the Ct value. Most samples did not rise above the threshold value; therefore, no comparisons could be made. However,  the cDNA samples and the ZMS2 RNA sample of the ACT1 gene did rise above the threshold. Although, these values were very high, which demonstrates that very small quantities of cDNA were produced. The Ct values of the ZMS2 cDNA and the wildtype cDNA were 26.5 and 33.9 respectively (Table1). The small cDNA quantities of the samples is abnormal for the ACT1 gene because it is a housekeeping gene. Beta-actin is a protein necessary for the function of yeast cells and it is needed in large quantities (Botstein et al., 1997; Bi et al., 1998). Therefore, the low amounts of cDNA detected by the RT-PCR illustrates that the initial quantity of cDNA used for the procedure was insufficient. This may explain why there was no detection of ALD6 cDNA. Since this gene is only necessary during glucose fermentation (Eglinton et al., 2002 and Saint-Prix et al. 2004), the quantities needed in the cell at any given time would be small compared to housekeeping genes such as ACT1. Therefore, the initial quantity of cDNA used for RT-PCR was extremely to small for the instrument to detect any cDNA for this gene. However, RT-PCR does differentiate which yeast, ZMS2 or wildtype, expressed more mRNA for the ACT1 gne. The results reveal that more ACT1 mRNA was expressed in the mutated yeast then the wildtype yeast. This correlates with the data obtained from the microarray experiment. The log2 fold change of the ACT1 gene is -0.792 (Table 2), which represents a slight decrease in expression of the ACT1 gene in the mutated yeast cells from the wildtype. However, both the RT-PCR and microarray data is not certain. The changes can not be proved to be significant in both cases; therefore, replicates of this experiment with a greater amount of initial cDNA must be done to determine significant changes in expression. A Ct value of 28.7 was determined for  the ACT1 ZMS2 RNA sample. This demonstrated that there may have been a DNA contamination in this sample. Theoretically, all RNA samples should have had no Ct values if they were pure RNA. Overall, a significant comparison of the two techniques is not possible with this data alone.           

                                               ACT1                                                                                   

                                                ALD6

Figure 2. PCR quantitation graph of ACT1 and ALD6 PCR samples. PCR samples were obtained from RNA extracted from mutated ZMS2 and wildtype Saccharomyces cerevisiae . Samples contained 3.1ul of RNA or cDNA and the control was a water blank. Cycle number is displayed on the x-axis and quantum yield (fluorescence) on the y-axis. The black dashed line right above zero is the threshold value used to find Ct. The Ct values were read at 80°C.
 

Table 1. Ct values for the RT-PCR products of the genes ACT1 and ALD6 of mutated ZMS2 and wildtype Saccharomyces cerevisiae. Samples contained 3.1ul of RNA or cDNA and the control was a water blank.  The Ct values were read at 80°C.

               ACT 1                                               ALD6

Table 2. Log2 fold change of the genes ACT1 and ALD6. The Log2 data represents the average expression of the genes ACT1 and ALD6 from the results of two microarray slides (green ZMS2 and red ZMS2) hybridized with cDNA of mutated ZMS2 and wildtype Saccharomyces cerevisiae.  

References:

Bi, E. et al. (1998) Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis. Journal of Cell Biology 142(5):1301-1312.

Botstein, D. et al. (1997) "The yeast cytoskeleton." Pp. 1-90 in The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology, edited by Pringle JR, Broach JR and Jones EW. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press

Eglinton, J. et al. (2002) Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene. Yeast 19(4):295-301

Nailis, H. et al. (2006) Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR. Molecular Biology  7(25):1186/1471-2199-7-25. 

Saint-Prix, F. et al. (2004) Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiology 150(7):2209-2220.

Zhang, Q. et al. (200) Cloning and Functional Analysis of cDNAs with Open Reading Frames for 300 Previously Undefined Genes Expressed in CD34+ Hematopoietic Stem/Progenitor Cells. Genome Research 10(10):1546-1560