Lab 06 - Quantitative Detection of Gene Expression Part II: Classical and Modern Strategies Flashcards
What is the enzyme which produces monoterpenes?
Terpene Synthases (Linalool Synthase)
Three most important questions asked about gene expression?
1) in what tissue is a particular gene expressed?
2) What is the relative expression of a given gene in various tissues?
3) How do various factors (temperature, starvation, presence of a toxin, etc.) affect the expression of a given gene?
What are the steps in a Nuclease Protection Assay?
1) Radio-labelled RNA or DNA probe complementary to the gene (mRNA) of interest (target RNA) is produced
2) The probe is hybridized to the target RNA.
3) The sample is then treated with enzymes (ribonucleases) that degrade single-stranded RNA, but not double-stranded RNA or DNA:RNA hybrid molecules
4) After the digestion of single stranded RNA and leftover probes, the remaining double-stranded probe: RNA hybrid molecules are precipitated out
5) They are then resolved on a denaturing polyacrylamide gel, and visualized by autoradiography or other suitable detection technologies.
What is the strength of the signal in a Nuclease Protection Assay proportional to? What can this knowledge be used for?
It is proportional to the quantity of the probe:RNA hybrid and can be used to determine the concentration of the target RNA in the original sample
How is Northern Blotting used to find the content of mRNA in the original sample?
1) RNA is resolved on an agarose gel by electrophoresis, and then transferred to a nylon membrane
2) A labeled probe that is specific to a given gene (target gene) is added to the membrane
3) The membrane is exposed to an X-ray film to visualize the signal of radiation given off by the bound probe
The intensity of the signal is proportional to the amount of the probe-mRNA hybrid, which is a measure of the quantity of mRNA present in the original sample.
Why is Real-Time PCR becoming the preferred method of quantifying mRNA?
Less Tedious
Smaller Sample Sizes
Steps in RT-PCR?
1) Total RNA or mRNA is reverse transcribed to cDNA
2) Target gene is amplified using complementary primers and other reagents (PCR)
3)
What is true of the concentration of DNA in reverse transcription if the reaction is stopped before the reagents run out?
The concentration of cDNA will be proportional to the initial concentration of the target gen in the sample.
Why is an internal standard used when running a RT-PCR?
An internal standard (gene expressed in equal mounts in ALL cells) is used because when comparing a target gene in two different samples, the sample amount of the template DNA must be added to the PCR reaction which is difficult to do. By using an internal standard you are giving a method to compare the actual concentration by using the internal standard to alter your results to resemble what would have happened if you had the same amount of template DNA.
What are the two most commonly used methods of RT-PCR?
rtPCR and qPCR (Both are suitable for studying small numbers of genes)
How rtPCR used to quantify the target gene in the original RNA sample?
The PCR reaction is terminated during the exponential phase of amplification and then run on an agarose gel (bands with a ladder) to determine the quantity.
How qPCR used to quantify the target gene in the original RNA sample?
A labelled probe or fluorescent dye is used during PCR to detect dsDNA at the end of each cycle. The data is stored and analyzed later so there is no need to stop the reaction
What is a microarray often used for?
Monitoring the expression of a large number of genes.
What does microarray technology depend on?
The process of specific probes hybridizing to their corresponding RNA or DNA template.
What is the level of hybridization of a microarray proportional to when studying mRNA?
Template present in the original sample, for mRNA specifically it represents the Gene expression from the original sample
