In humans, congenital heart disease is a common birth defect that affects approximately 1 out of 125 live births. Using reverse transcription PCR (RT-PCR) Samir Zaidi and colleagues [(2013) Nature 498:220.223] determined that approximately 10 percent of the cases resulted from point mutations, often involving histone function. To capture products of gene expression in developing hearts, they used oligo(dT) in their reverse transcription protocol.

If one were interested in comparing the quantitative distribution of gene expression in say, the right and left side of a developing heart, how might one proceed using RT-PCR?

Verified step by step guidance

1. <span>Step 1: Collect tissue samples from both the right and left sides of the developing heart. Ensure that the samples are collected under similar conditions to minimize variability.</span>
2. <span>Step 2: Isolate total RNA from each of the tissue samples. This involves lysing the cells and using a method such as column purification or phenol-chloroform extraction to obtain the RNA.</span>
3. <span>Step 3: Use oligo(dT) primers in the reverse transcription step to synthesize cDNA from the mRNA present in the samples. Oligo(dT) primers will bind to the poly-A tail of mRNA, ensuring that only mRNA is reverse transcribed.</span>
4. <span>Step 4: Perform quantitative PCR (qPCR) on the cDNA using gene-specific primers. This will allow you to quantify the expression levels of specific genes of interest in both the right and left heart samples.</span>
5. <span>Step 5: Analyze the qPCR data to compare the expression levels of the genes between the right and left sides of the heart. Use appropriate statistical methods to determine if there are significant differences in gene expression.</span>

Key Concepts

Reverse Transcription PCR (RT-PCR)

RT-PCR is a laboratory technique used to convert RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase. This process allows researchers to amplify specific RNA sequences, making it easier to study gene expression levels. In the context of comparing gene expression in different tissues, RT-PCR can quantify the amount of cDNA produced from RNA samples, providing insights into the relative expression of genes in those tissues.

Gene Expression Analysis

Gene expression analysis involves measuring the activity of genes in a given cell or tissue type, which can reveal how genes are regulated and their functional roles. By comparing gene expression levels between the right and left sides of a developing heart, researchers can identify differences in gene activity that may contribute to the development of congenital heart disease. This analysis often employs quantitative methods, such as RT-PCR, to provide precise measurements of gene expression.

Quantitative Comparison

Quantitative comparison in gene expression studies refers to the process of measuring and comparing the levels of specific mRNA transcripts between different samples. This can be achieved using techniques like RT-PCR, where the amount of amplified cDNA is quantified to determine expression levels. By establishing a baseline or control, researchers can assess how gene expression varies between the right and left sides of the heart, leading to insights into developmental processes and potential abnormalities.