Reverse transcriptase polymerase chain reaction (RT-PCR) of a UV-dependent gene

When studying DNA, researchers often amplify regions of interest using the polymerase chain reaction (PCR). PCR replicates specific fragments between two primers, which are complementary to short segments of DNA.

Researchers can study gene transcription using reverse transcriptase PCR (RT-PCR), which involves exploiting the ability of reverse transcriptase to synthesize complementary DNA (cDNA) from mRNA transcripts, and then using PCR to amplify regions of interest (Figure 1). An important step in RT-PCR is the purification of mRNA from the cell extracts. Without this purification step, genomic DNA can contaminate the PCR reaction and become amplified along with mRNA. Like other DNA polymerases, reverse transcriptase requires a primer. Researchers must take care not to contaminate their RNA samples with widely prevalent RNAse enzymes, which degrade RNA and can ruin experiments. Inhibitors of RNAse can be used to prevent such degradation in the case of RNAse contamination.

A graduate student interested in skin cancer and melanin synthesis used RT-PCR to measure how UV light exposure stimulated production of melanin. Specifically, she wanted to determine the effect of UV light on the transcription of the gene for pro-opiomelanocortin (POMC) in keratinocytes. As a post-translational modification, POMC is cleaved to yield multiple active peptides, including melanocyte-stimulating hormone ($\alpha$‍-MSH), which can then trigger synthesis of melanin in nearby melanocytes.

Figure 1: Schematic of RT-PCR experiment. The position of the reverse transcriptase primer (Primer 1) and the positions of the PCR primers (Primers A and B) are shown.

The student exposed keratinocytes to ultraviolet light of a constant intensity for various lengths of time. After this UV exposure, she harvested cells and conducted RT-PCR, using primers specific for a fragment of POMC spanning an intron (Figure 1). She then loaded her cDNA samples into an agarose gel and applied voltage to separate the fragments by size. The brightness of the bands (Figure 2) represents the quantity of amplified cDNA present in the gel for that sample, and therefore the quantity of POMC mRNA present in the original sample.

To determine size of her fragments, the student added a DNA ladder to the leftmost lane, which included $8$‍ DNA fragments at $100$‍ base pair intervals ranging from $100$‍ to $800$‍ base pairs. She also included positive and negative controls.

Figure 2: Gel electrophoresis. Samples added at “top” of gel in the photo below. Lanes 1-5 contain RT-PCR products from keratinocytes exposed to different durations of UV exposure. Lane 6 = positive control; Lane 7 = negative control; Ladder = 8 DNA fragments ranging from 100 to 800 base pairs in length.