Analyze the expression levels of the MC1R and GAPDH genes in human biological samples using polymerase chain reaction (PCR) techniques: Molecular Biology, Thesis, NIH, Ireland

Aim:

The primary aim of this experiment is to quantitatively analyse the expression levels of the MC1R and GAPDH genes in human biological samples using polymerase chain reaction (PCR) techniques

Objective:

To extract and purify total RNA: Utilize a robust RNA purification protocol to extract high-quality RNA from human saliva and swab samples. Validate the integrity and purity of the extracted RNA to ensure it meets the standards for reverse transcription.

To assess RNA quality and concentration: Employ a microvolume spectrophotometer to measure the concentration of RNA. Determine the purity of RNA samples by calculating the A260/A280 ratio, ensuring they are free from protein contamination.

To synthesize complementary DNA (cDNA):Reverse transcribe the purified RNA into cDNA, which will serve as a template for subsequent PCR amplification.

To amplify target gene sequences: Design and utilize specific primers for both MC1R and GAPDH genes to amplify target regions accurately. Perform PCR to amplify the cDNA of the target genes, enabling quantitative analysis.

To quantify gene expression levels: Analyse the amplified DNA products to determine the expression levels of MC1R and GAPDH genes. Compare the relative expression of MC1R in different samples and correlate with phenotypic data if available.

To ensure experimental accuracy and reproducibility: Conduct all experiments in triplicate to validate the reproducibility of the results. Use GAPDH gene expression as an internal control to normalize the data and facilitate accurate comparison across samples.

To interpret and report findings: Assess and compare the expression patterns of the MC1R and GAPDH genes in the collected samples. Discuss the potential implications of the expression levels in relation to pigmentation and cellular metabolic status.