Bovine Serum Albumin Reverses Inhibition of RT-PCR by Melanin

Melanin contained in pigment cells in a variety of tissues co-purifies with nucleic acids in standard RNA (or DNA) extraction procedures. The presence of melanin in RNA (or DNA) templates can inhibit reverse transcription polymerase chain reactions (RT-PCR) and/or PCR (5). In the past, to circumvent this problem, melanin has been separated from DNA by either columnchromatography (5) or acid-precipitation (3). In this study, a facile approach is described to overcome melanin inhibition of RT-PCR. Specifically, the addition of >15 μg of bovine serum albumin (BSA) per 25 μL RT-PCR mixture is shown to reverse melanin inhibition (Figures 2, A and B). BSA has been used previously to prevent inhibition of PCR amplifications by hemin, iron chloride, tannic acids, fulvic acids, extracts from feces, freshwater or marine water (4). In this study, we describe the doseresponse effect of melanin on RT-PCR inhibition and the effect of BSA in overcoming melanin inhibition. The addition of 2 μg of synthetic melanin (Catalog No. M-8631; Sigma Chemical, St. Louis, MO, USA) to a 25-μL RT-PCR can completely inhibit RTPCR of aldolase mRNA (Figure 1A). A similar degree of inhibition was observed when melanin-containing RNA preparations were prepared from either normal melanocyte cell strains (Figure 2A) or melanoma cell lines (data not shown). While RT-PCR of 0.5 μg total RNA will normally yield a product after 16 cycles of PCR following RT (2), when 2 μg of melanin were present, no RT-PCR product was obtained after 45 cycles of PCR. The addition of >15 μg BSA to the reaction mixture alleviated melanin inhibition and allowed detection of a PCR product by PCR cycle 16. The addition of 20–23 μg BSA per assay appeared to be optimum (Figure 1B) in overcoming melanin inhibition. Fatty acid-free BSA (Catalog No. A7511; Sigma Chemical), >97% pure alcohol-precipitated BSA (Catalog No. A-4378; Sigma Chemical) and Fraction V 98%–99%-pure BSA (Catalog No. A-7906; Sigma Chemical) were found to be equally effective in reversing melanin-mediated RT-PCR inhibition (data not shown). It was reported previously that melanin inhibits PCR (5). We found that melanin slightly inhibits the RT step as well. This determination was made as follows: (i) two RT reactions were carried out: a control and an RT containing 2 μg of synthetic melanin, and (ii) PCR was carried out on 1/100 dilution of the RT-generated cDNAs. Note that 1/100 dilution was used as a means to eliminate the effects of melanin at the PCR step. A small reduction in PCR product was noted after 2 μg of synthetic melanin were added at the RT step. This amount of melanin present at the PCR step would have been completely inhibitory. Thus, melanin appears to have only a small effect on reverse transcription. In summary, the simple addition of >15 μg BSA per 25 μL RT-PCR mixture effectively overcomes inhibition of RT-PCR by melanin. Addition of up to 23 μg BSA per RT-PCR appeared to have no deleterious effects on RT-PCR product yield. This modified procedure should allow more effective RT-PCR analysis in melanin-containing cells or tissues (e.g., skin, hair or eyes).