Question

Suppose you want to determine if excessive lipid ingestion alters gene transcription. Describe and explain all the steps you would carry out to do a microarray-based study of the question. Include your choice of subjects and tissues to be investigated. (The statement of the problem to be studied- effects of "excessive lipid ingestion"-is deliberately vague, and you should refine it by specifying and explaining an operational procedure.)

Short answer

To explore the effects of excessive lipid ingestion on gene transcription, it is required to use microarray experiment. The study would involve feeding mice with a normal diet and high-fat diet. Then, RNA is extracted from liver tissue to perform the microarray experiment. The output from the microarray is analyzed to interpret the differences of gene transcription between the two groups.

Step-by-step solution

Refine the problem

Firstly, it is important to refine the problem statement. Instead of just 'excessive lipid ingestion', a more specific and measurable variable should be defined - e.g. ingestion of a high-fat diet for a certain period.

Selection of Subjects

Next, the appropriate subjects need to be chosen. Animal models like lab mice are often used in such studies due to ethical reasons and ease of control over diet. The mice could be divided into a control group fed a normal diet and an experimental group fed a high-fat diet.

Selection of Tissues

Choosing the right tissue is crucial. Since the lipid metabolism primarily happens in the liver, taking a liver tissue sample would be an appropriate choice.

Sample Preparation

Next, RNA is extracted from the liver tissues. This RNA represents the active transcription at the time.

Microarray Experiment

The RNA from each group is then converted into a differently-colored surrogate cDNA, and are then poured simultaneously onto a microarray. Each spot on the microarray has DNA fragments from one specific gene. If a gene is being transcribed, its corresponding mRNA will bind to its cDNA on the chip and will be recorded by measuring the fluorescence.

Data Interpretation

The data from the microarray is then interpreted. The intensity of the colors in the microarray spots represent the level of gene transcription. Comparing the spot intensities between high-fat and normal diet groups can identify transcription changes due to excessive lipid ingestion. Bioinformatics tools can help analysing and visualizing this data.

Key concepts

Lipid Metabolism

Lipid metabolism encompasses the complex processes of how the body uses and stores fats. Our tissues can break down lipids to release energy, and synthesizing or storing them when we have excess calories. The liver plays a pivotal role in these metabolic pathways.

To understand the impact of lipid ingestion on gene transcription, scientists can examine how a high-fat diet alters the activity of genes related to lipid metabolism. This involves not only looking at the end products, like cholesterol or triglycerides, but also at how expression levels of various enzymes and proteins involved in lipid processing change. These insights can reveal potential targets for treating metabolic disorders.

High-Fat Diet Effects

A high-fat diet is known to influence our metabolic health, potentially leading to obesity, insulin resistance, and other chronic conditions, if sustained over time. When designing experiments to evaluate the effects of such a diet, it's essential to characterize the diet precisely, including the type, quantity, and duration of fat consumed.

Understanding these effects at the molecular level involves examining the gene transcription profiles in response to the diet. For instance, a high-fat diet might upregulate genes involved in fat storage or downregulate those involved in lipid oxidation. It's the subtle shifts in these patterns that the microarray-based gene transcription studies aim to uncover.

RNA Extraction

RNA extraction is a crucial step in assessing gene transcription, as RNA levels directly reflect gene activity. This process involves isolating the RNA from cells or tissues whilst protecting it from enzymes that might degrade it.

Once extracted, the quality and quantity of RNA can be assessed, which is important because only high-quality RNA can provide reliable data in subsequent steps, such as conversion to cDNA and hybridization during a microarray assay. Detailed protocols with specific reagents ensure that RNA of sufficient purity is obtained for accurate fluorescence measurement.

Fluorescence Measurement

After isolating and preparing RNA, it is converted to cDNA, which can be labeled with fluorescent dyes. These labels allow scientists to measure the abundance of different mRNA transcripts via fluorescence measurement in a microarray analysis.

By exposing a microarray to cDNA from different samples, hybridization occurs between complementary DNA strands. The intensity of the emitted fluorescence at each microarray spot correlates with the amount of mRNA bound there, hence the level of gene transcription. The comparison of fluorescence between different conditions, like a high-fat diet versus a normal diet, can thus reveal the effects of dietary changes on gene expression.