Measuring Gene Expression (#13)

Question 1

Microarray uses

Answer 1

Used to compare two conditions. Examples:
1. Normal vs. cancerous tissue
2. Wild type vs. transgenic organism
3. Before and after drug treatment
4. Comparison of different cell lines
Uses:
1. Basic research - gene expression profiling and gene discovery
2. Medical research - pharmacology/toxicology research; disease diagnosis/prognosis

Question 2

Quantitative measurement of expression

Answer 2

The most accurate method for measuring gene expression is real time PCR
Differences from traditional PCR:
1. Results of traditional PCR must be observed on a gel, whereas real time PCR results are observed in real time (after every cycle) using a computer
2. The reason that real time PCR can be measured as the reaction is proceeding is due to the fluorescent chemistries employed in real time PCR

Question 3

Two types of qPCR

Answer 3

1. Hydrolysis (TaqMan) probe
   a. A fluorescently labeled probe is annealed between the PCR primers
   b. Upon DNA synthesis, the probe is hydrolyzed and released from the DNA
   c. This results in detectable fluorescence indicative of how much DNA was amplified
2. SYBR Green dye
   a. SYBR Green is an intercalating agent that when bound to DNA, results in green fluorescence
   b. The more DNA synthesized, the more SYBR Green fluorescence that will be detected

Question 4

TaqMan probe qPCR

Answer 4

1. Polymerization - A fluorescent reporter dye and a quencher are attached to the 5’ and 3’ ends of TaqMan probe
2. Strand displacement - When the probe is intact, the reporter dye emission is quenched
3. Cleavage - During each extension cycle, the DNA polymerase cleaves the reporter dye from the probe.
4. Polymerization complete - Once separated from the quencher, the reporter dye emits its characteristic fluorescence

Question 5

SYBR Green qPCR

Answer 5

1. Reaction setup - The SYBR Green Dye fluorescences when bound to double stranded DNA
2. Denaturation - When DNA is denatured, the SYBR Green Dye is released and the fluorescence is drastically reduced
3. Polymerization - During extension, primers anneal and PCR product is generated
4. Polymerization completed - When polymerization is complete, SYBR Green Dye binds to the double stranded product, resulting in a net increase in fluorescence by the 7900HT system

Question 6

Dissociation curve to determine purity of product

Answer 6

Dissociation curves are only applicable when using SYBR Green Dye. Remember: ANY DNA including nonspecific products (primer dimers, will be detected by SYBR Green
Increasing temp will cause DNA strands to denature. Nonspecific products will denature at different (typically lower) temps that the desired product

Question 7

Two types of quantitation methods

Answer 7

1. Absolute - quantitates the samples based on the amplification profiles of samples with known amounts
2. Relative - quantitates the samples compared to a reference sample of unknown amounts. This type of analysis is often used to determine gene expression changes between two different conditions.