Practice Exam 3 (RNA Techniques) 1. You are interested in whether or not Dr. Rob
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Practice Exam 3 (RNA Techniques) 1. You are interested in whether or not Dr. Robertson's biotech class is so stimulating that it induces (turns up) the expression of neuronal growth factor (NGF) in the brain of biotech students. Since all the students in the class are convinced their brains will get bigger as a result of Dr. Robertson's stimulating lectures they agree to allow you to biopsy enough brain material both before taking the class and after taking the class to do northern blots. You prep the tissues carefully and isolate total RNA from each sample a) What precautions must you taken when working with RNA in general? b) Once you have collected total RNA from brain tissue of 3 students in the class, you do the northern blots by running the before and after samples on a gel, transferring the separated RNAs onto nitrocellulose, blocking the blot, and probing with a probe specific for NGF. What was special about the gel you used? c) Once you visualize the probe from your northern blot you get the results shown below. What can you conclude from these results about the expression of NGF in response to taking Dr. Robertson's biotech class? Northern Blot before #2 #3 after #213 #1 #1 4.0 3.0 2.0 .0 0.5Explanation / Answer
Answer-a: Following precautions must be taken to handle RNA samples-
1. RNA is very sensitive to higher temperature. They are prone to degradation at temperatures higher than 65 degree celcius. So, the RN samples must be stored freezing temperatures, otherwise, it would destroy the integrity of the RNA to be analyzed.
2. While working with RNA or its sample collection, one must always wear gloves. Sample must not be treated with the bare hands because our hands also contain a film of nucleases which would destroy the sample. The surrounding areas must also be wiped with the 100% ethanol.
3. All the glasswares, instruments and plasticwares must be properly autoclaved and treated with the RNAase inactivating substances.
4. Sample must be extracted as quickly as possible because RNA is very sensitive and might get destroyed at room temperature.
5. RNA samples must be stored at -70 degrees as it remain stable at this point. Before using it, it must be thawed at 37 degree.
Answer-b: For performing the Northern blotting, Agarose gel was used while performing the electrophoresis. Agarose gel is favoured in this procedure, because it has an ability to readily form hydrogen bonds when it is dissolved and heated along with the buffer. It polymerises quickly. Moreover, it does not require any additional catalyst which aids in its polymerization. This gel is comparable safe to handle because it is non-toxic. Sample can be easily recoved when required, by treating it with certain chemicals which would dgest the gel and separates out the sample.
Answer-c: Bands are obtained in the gel when numerous small fragments of almost equal size and weight settles down or over-lap in the gel pores. If we compare the bands of those samples taken before lecture, then we observe that they are slight thin. On the other hand, if we see the bands of sample taken after the lecture, they happen to be thicker. They are thicker because the number of fragments of RNA are pretty more than the sample for RNA taken before lecture. Large number of RNA fragments making thick band given us the direct conclusion that Dr.Robertson's lecture does stimulates the NGF which would differentiate more neurons and differentiated neuron would inturn produce the RNA and subsequent necessary proteins. Thinner band indicates the less or no stimulating work by NGF, which therefore produced low RNA in neurons.
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