Flashcards in Lab 06 - Quantitative Detection of Gene Expression Part II: Classical and Modern Strategies Deck (33):
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?
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)
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
Unlike Western Blotting, how is the probe used in Microarrays?
In microarrays the probes are hybridized to the surface of the chip and the chip is exposed to the labelled template (Western = Template is hybridized to membrane)
How is the signal of the Microarray detected
Red and Green laser light for different treatments
What are the two methods for generating Microarray Probes?
1) cDNA spotted microarrays: a robot spotter spots (loads) the chip (or the microarray) with cDNA or small PCR-amplified DNA fragments corresponding to specific genes
2) oligonucleotide microarrays: short DNA oligonucleotides (probes) are spotted (or directly synthesized) on the array (advantage: more than one probe per gene can be used, ~n= 4, maximizing specificity of the analysis)
How can we identify the expression profile of a sample?
Observe all spots microarray at the same time
Steps of a Microarray?
1. Probes are generated against all of the known mRNA species from the sample being studied.
2. Probes are loaded onto a gene chip. Note that probes can be synthesized directly on the chip.
3. Messenger RNA is extracted from both samples, and reverse transcribed to produce cDNA. The cDNA from one sample is labeled with a Red dye and that from the other sample with a different (green) dye.
4. Both cDNA samples are mixed and applied onto the chip, and allowed to hybridize.
5. Excess cDNA is washed off, and signals emitted from the two dyes are detected and quantified using scanners. The data is analyzed to identify deferentially expressed genes.
Why are microarrays generally not used?
Performing a microarray assay is very time consuming, expensive, and requires specialized equipment but it is good for looking at the expression of multiple genes at once
What is the threshold in qPCR? What causes it to vary?
1) The minimum accepted fluorescence level
2) The dye used
What is background noise in qPCR? When is this mainly a problem?
1) Fluorescence emitted below the threshold level
2) In early amplification because unbound dye emits small amounts of fluorescence and dye molecules can bind each other creating fluorescence
What is the Cycle Threshold Value (CT Value)? When does this typically occur?
1) The number of cycle required for the fluorescence emitted to pass the threshold value
2) At the beginning of the exponential phase
What is a calibrator / control in qPCR?
A sample used to compare expression of the target gene (we used leaf)
Unknown genes who's expression is to be analysed
What is No-Template Control in qPCR?
What should not take place in this control and if it does what does that indicate?
1) Reaction mix devoid of template used to establish minimal or no primer-dimer contribution to the emitted fluorescence.
2) No DNA amplification should take place in this control. Any amplification is an indication of contamination
What is a Non-Reverse Transcribed Control in qPCR?
Degraded genomic DNA used to ensure no false positive from genomic DNA contamination
What does qPCR require in terms of equipment?
1) Thermal Cycler
2) Fluorescent Detector
3) Computer to save and analyze data
What is the main difference between qPCR and rtPCR?
The time of data analysis
What are some common probes in qPCR?
1) Molecular Beacons
4) Variants of SYBR Green (Dye)
What do we use from qPCR to calculate the absolute initial concentration of the target mRNA in the sample?
The CT values (For both absolute and relative between tissues)