1. Methods for
Gene Activity
Analysis By
Auni Hovanesian
Krista Templeton

2. What Methods Can We Use to Study Gene Expression?
Three Basic Approaches:
RT-PCR
GeneChip Microarray
Upstream Regulatory Region Analysis

3. What Is The Basis of RT-PCR Activity Analysis?
mRNA!
mRNA is only present in cells for genes that have been transcribed and will usually be actively expressed once translated into proteins
RT-PCR links gene expression to mRNA presence in different compartments of plant
But in order to trace mRNA back to an original gene, must amplify sample, since often only small amounts present

4. Why RT-PCR Rather Than Regular PCR?
mRNA will not work in our PCR reaction, so need to convert it to cDNA
reagents being used in PCR are DNA-specific (i.e. DNA polymerase)
DNA is more stable than RNA
More practical for long-term storage purposes
RNA’s instability would give lower yield in PCR

5. Reverse Transcriptase
So How Does RT-PCR Work?
Isolate mRNA from area-specific plant tissue/organ samples
Convert all isolated mRNA strands to cDNA using Reverse Transcriptase
mRNA
AAAAAAA
Reverse Transcriptase
cDNA

6. 3) Use first cDNA template as now compatible basis of PCR:
Only need to synthesize a single strand of cDNA template to start PCR
One cDNA representing every mRNA in each sample--like gene expression library
PCR is like selection of a particular gene from the library (if it is there!)
Gene-specific primers designed in exons (due mRNA splicing)
If PCR product formed with gene-specific primers for RT-PCR, will have amplified the cDNA correlate of original gene
Indicates presence of mRNA correlating to gene of interest
Helps localize gene activity to wherever sample came from

7. Sample RT-PCR Results 100 bp Silique RT+ Silique RT- Positive Control
Negative Control
Leaf RT+
Leaf RT-
Tubulin Bands
475 bp
Hypothetical cDNA Band
200 bp

8. So How Do We Verify and Further Specify RT-PCR Results?
GeneChip Microarray Analysis!

9. What is a GeneChip Microarray?
Using cDNA created from the mRNA isolated from various organs, we can analyze the mRNA accumulation levels for all genes
Done by creating the complementary strands of all the known gene sequences and assembling them on a chip
Different chips are used for various stages of development
The cDNA sequences are tagged with flourescent labels that glow a certain color when in contact with the complementary strand; colors read by a computer

10. What is the Resulting Image?
Red means active
Green means not as active
No color means no activity

11. What Does the Online GeneChip Report Look Like?

12. So, How Do RT-PCR and GeneChips Complement Each Other?
RT-PCR may be slightly more accurate, since working with smaller fragments (only portions of cDNA)
But GeneChips provide more specificity more efficiently
The two combined provide convenient checkpoints for each other’s results
both working with same principle of mRNA level analysis to determine gene expression

13. What Is Another Method of Gene Expression Analysis?

14. Upstream Region Analysis

15. Overview
The upstream regulatory region of a gene contains its “on” switch.
Once the upstream region is fused to GFP (green fluorescent protein) or GUS (betaglucuronidase) it can be transformed into the Arabidopsis plant
Once transformation has occurred gene expression will indicate where the gene of interest is transcribed.

16. Strategy of Promoter Activity Analysis
Arabidopsis Genomic DNA
PCR amplification of upstream region
With Gene-specific Primers
And High Fidelity DNA Polymerase
PCR Product
pENTR/D-TOPO vector
Ligation: Population of Recombinant Plasmid (vector+PCR product) and NON-recombinant plasmid (vectory only)
Transformation of competent E.coli cells
Screening for E.coli cells harboring recombinant plasmid
Confirmed Recombinant plasmid DNA: Verifying the authenticity of recombinant plasmid DNA by Restriction Enzyme Digestion
Recombinant Plasmid DNA + Beta-Gluronidase (GUS) gene carrying T-DNA Vector
DNA sequences: verification of the cloned Promoter Region by Sequencing Analysis.
Sequence Analysis and confirmed identity of the cloned upstream region

17. What Are the Steps Required to Isolate the Promoter Region?
Conduct PCR on the Arabidopsis DNA in order to amplify the upstream region with iProof polymerase

18. Why Use a Proofreading Polymerase?
iProof polymerase corrects nucleotide errors during amplification
One mutation could affect the transcription of a gene

19. What To Do After the Promoter Region Has Been Ampified
The section needs to be enzymatically inserted into a plasmid vector where it can be placed into an E.coli cell.
DNA
pENTR-TOPO vector
Recombinant Plasmid
Mix E.coli cells with plasmids in presence of CaCl. Culture on nutrient agar plates containing ampicillin.

20. What Does Topoisomerase 1 Do?
The pENTR/d-TOPO vector contains Topoisomerase 1
Relieves supercoils in circular DNA plasmids by nicking one of the strands of the DNA double helix
Linearized the pENTR vector, allowing insertion of the PCR fragment
Re-ligates vector

21. How Do GUS and GFP Work and What Are Their Differences?
A mature Arabidopsis embryo expressing Green Fluorescent Protein
GUS is the more sensitive of the two Common Reporter Genes
GUS and GFP connect to the promoter region. When the genes are turned on through the promoter region GUS and GFP are turned on also

22. Acknowledgements
Jordan, Jennifer, and Brian from Spring 2006 for some useful slide ideas and diagrams
Kelli for her presentation on Upstream Region Analysis
Brandon for all his tech support!
Anhthu, Bekah, and Daisy for their help and many explanations