Question

Scientists studying Alzheimer’s have found that patients who have Alzheimer’s have higher levels of tau protein accumulation on their frontal cortex. In order to prove causation, a researcher uses real time PCR. How would you use real time PCR to do the same study?

Answer 1

Real time PCR are used with reverse transcription method to study the expression of the tau protein. Real time allows the quantification of tau gene even if the it's amount is too low. This is possible due to application of tau gene with fluroscent technology. This process begin with RNA extraction followed by cDNA preparation using reverse transcriptase and lastly PCR. So, as cDNA do not have any introns sequence,, it expression analysis give the expression of Tau protein.

Answer 2

To study the potential causation between tau protein accumulation and Alzheimer's disease using real-time PCR (polymerase chain reaction), the researcher would need to follow specific steps to analyze gene expression levels in the frontal cortex of patients with Alzheimer's and compare them with control samples. Real-time PCR is a powerful technique for quantifying gene expression levels and detecting changes in specific gene targets. Here's how the researcher could conduct the study:

1. Sample Collection: Collect tissue samples from the frontal cortex of patients diagnosed with Alzheimer's disease and age-matched control individuals without Alzheimer's. Ensure proper sample preservation and storage to maintain RNA integrity.

2. RNA Extraction: Isolate total RNA from the tissue samples using a suitable RNA extraction method. High-quality RNA is crucial for accurate real-time PCR results.

3. cDNA Synthesis: Reverse transcribe the isolated RNA into complementary DNA (cDNA) using reverse transcriptase enzyme and specific primers. This step converts the RNA molecules into a stable cDNA form for PCR analysis.

4. Primer Design: Design specific PCR primers for the gene encoding tau protein and a reference gene (housekeeping gene) that shows stable expression across all samples. The reference gene will be used to normalize the target gene's expression levels.

5. Real-time PCR: Perform real-time PCR using a thermal cycler equipped with a fluorescent detection system. This technique allows the researcher to monitor the PCR amplification in real-time as it progresses.

6. Data Analysis: Analyze the real-time PCR data using appropriate software. Calculate the relative gene expression levels of tau protein in Alzheimer's patients and control samples by comparing the cycle threshold (Ct) values of the target gene with the reference gene (using the ΔΔCt method).

7. Statistical Analysis: Perform statistical analysis to compare the expression levels of tau protein between Alzheimer's patients and control samples. This analysis will help determine if there is a significant difference in tau protein accumulation between the two groups.

8. Interpretation: Interpret the results to determine if there is a significant correlation between tau protein accumulation and Alzheimer's disease. If the real-time PCR analysis shows higher levels of tau protein in the frontal cortex of Alzheimer's patients compared to controls, it suggests a potential causative relationship between tau accumulation and the disease.

It's essential to conduct the study with appropriate controls and replicate experiments to ensure the reliability of the results. Real-time PCR is a valuable tool for investigating gene expression changes and can provide valuable insights into the molecular mechanisms underlying diseases like Alzheimer's.