Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove
Click here for the English version

Biology

微阵列分析FOR酿酒酵母 Published: April 7, 2011 doi: 10.3791/2585

Summary

在这个协议中,在酵母中的基因表达(

Abstract

在这个协议中,酵母( 酿酒酵母 )中的基因表达改变曝光后添加过氧化氢 ​​(H 2 O 2),氧化剂诱导的氧化应激。在实验过程中,酵母是在1/2X YPD 3X葡萄糖肉汤培养48小时。文化是分割成一个控制和治疗组。实验文化是用0.5毫米的H 2 O 2汉克斯治疗的缓冲液(HBSS)1小时。控制文化是唯一的HBSS治疗。总RNA是从两种不同的文化提取和转换通过一个多步骤的过程中,以生物素标记的cRNA产品。最后的合成产品带回UVM芯片的核心设施,Affymetrix公司的酵母基因芯片杂交。由此产生的基因表达数据上传到生物信息学数据分析软件。

Protocol

Discussion

Disclosures

生产这种视频文章EMD - Millipore公司赞助。

Acknowledgments

Materials

Name Company Catalog Number Comments
Spetrophotometer
Thermocyler
Microcentrifuge
Vortex
Stir Plates (2)
P-10s pipettors
P-20's pipettors:
P-200's pipettors:
P-1000's pipettors:
Camera and transilluminator
E-Gel Apparatus Invitrogen G6000-08
E-Gels Invitrogen G6018-02
Axygen 1.7 ml. Clear Krackeler Scientific, Inc. 383-MCT175C
Axygen 0.5 ml clear tubes Krackeler Scientific, Inc. 383-MCT060C
Axygen 2ml capture tubes Krackeler Scientific, Inc. 383-MCT200NC
boxes of P-10 ART tips: CLP BT10XL
Boxes of P-100 ART tips: CLP BT200
Boxes of P-20 ART Tips: CLP BT20
Boxes of P-1000 ART Tips: CLP BT1000
Yeast Chips
25 ml sterile disposable pipets-
5ml sterile disposable pipets-
Microfuge tube racks
KimWipes:
Lab Coats
Stir Bars
Culture flasks
Innoculating loops
Sharpies
Gloves
Autoclave tape
Stirrer bars
30% Hydrogen Peroxide EMD Millipore 386790
HBSS without Ca, Mg, or dye Sigma-Aldrich H6648-100ml
Qiagen Rneasy Mini Kit: Qiagen 74104
Qiagen DNase Kit: Qiagen 79254
Rnase Remover Denville Scientific D1180
Harleco Alcohol 100% EMD Millipore 65347
ENZO IVT Kit Enzo Life Sciences ENZ-42655-10
Lyticase: one bottle VWR international IC19012310
Water, Cell Culture grade EMD Millipore 4.86505
Yeast culture: ATCC 18824
YPD broth powder EMD Millipore 4.8504
D(+) Glucose, Anhydrous EMD Millipore 346351
Sorbitol EMD Millipore 56755
EDTA EMD Millipore 4005
SDS solution, 20% EMD Millipore 7990-OP
Pellet Paint EMD Millipore 69049
PCI RNase DNase free (7 aliquots): Fisher Scientific AC32711-500
7.5M NH4OAc Fisher Scientific ICN1987 5980
Fragmentation Buffer (200 mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM MgOA)
Trizma Base Fiaher 50-899-90034
KOAc Fisher Scientific NC9757725
MgOA Fisher Scientific NC9681042
Loading Dye Invitrogen 10482055
PCR Markers EMD Millipore 69278-3
Phase Lock Gels Fisher Scientific FP2302820
One-Cycle cDNA Kit Invitrogen A10752-030
Includes;
E. coli DNA Pol
dNTP's
T4 DNA Pol.
T-7 oligod(T)
0.5ml EDTA pH 8.0
First Strand Buffer
E. Coli RNaseH
Second Strand Buffer
E. Coli DNA ligase
SuperScript II
DTT

DOWNLOAD MATERIALS LIST

References

  1. Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A. 87, 1663-1667 (1990).
  2. Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., Botstein, D., Brown, P. O. Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell. 11, 4241-4257 (2000).
  3. D'Autréaux, B., Toledano, M. B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol. 8, 813-824 (2007).
  4. Shenton, D., Smirnova, J. B., Selley, J. N., Carroll, K., Hubbard, S. J., Pavitt, G. D., Ashe, M. P., Grant, C. M. Global translational responses to oxidative stress impact upon multiple levels of protein synthesis. J Biol Chem. 281, 29011-29021 (2006).
  5. Godon, C., Lagniel, G., Lee, J., Buhler, J. M., Kieffer, S., Perrot, M., Boucherie, H., Toledano, M. B., Labarre, J. The H2O2 stimulon in Saccharomyces cerevisiae. J Biol Chem. 273, 22480-229 (1998).
  6. Vermont Genetics Network. Microarray Outreach. , (2008).
  7. Affymetrix GeneChip Expression Analysis: Technical Manual. , Affymetrix. Santa Clara, CA. (2004).
  8. Huang, D. W., Sherman, B. T., Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 4, 44-57 (2009).
  9. Dennis, G. Jr, Sherman, B. T., Hosack, D. A., Yang, J., Gao, W., Lane, H. C., Lempicki, R. A. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 4, P3-P3 (2003).

Tags

基本协议,50期的cRNA,基因芯片,基因,颗粒漆
微阵列分析FOR<em>酿酒酵母</em
Play Video
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Tighe, S., Hunter, T., Reed, P.,More

Tighe, S., Hunter, T., Reed, P., Murray, J. Microarray Analysis for Saccharomyces cerevisiae. J. Vis. Exp. (50), e2585, doi:10.3791/2585 (2011).

Less
Copy Citation Download Citation Reprints and Permissions
View Video
Waiting X
Simple Hit Counter