An improved method for RNA isolation and removal of melanin contamination from melanoma tissue: implications for tumor antigen detection and amplification.
Several kits are available to isolate RNA(1) from tissues. However, melanoma tissue is often rich in melanin that co-purifies with DNA/RNA and inhibits subsequent PCR reactions, hampering tumor antigen detection and amplification. This problem has not yet been addressed systematically. Here we generated a photometric protocol to determine both the melanin and RNA concentration by correcting the latter for the absorption coefficient of melanin at 260 nm. Subsequently, different combinations of silica-based RNA-binding, size-exclusion, and ion-exchange columns were used in 8 protocols for isolation of RNA from melanoma tissue to compare efficacy of melanin removal and yield of RNA. Furthermore, the capability of the different RNA preparations to function as template in RT-PCRs with products of different length, i.e. GAP-DH, tyrosinase, and gp100, was tested. We found that the combination of silica-based RNA-binding and size-exclusion columns was not sufficient to remove melanin from highly contaminated tumor samples, and subsequent RT-PCR failed to give larger products. However, protocols including ion-exchange columns resulted in efficient removal of melanin, while retaining reasonable RNA yields in samples from highly pigmented melanomas. Efficient RT-PCR of larger products turned out to be inversely correlated to the melanin contamination. This RNA-purification method will help scientists to isolate polynucleotides from melanin-containing tumor samples, which subsequently can be used in antigen detection assays and vaccination strategies using amplified total tumor RNA.
Dörrie
Jan
J
Department of Dermatology, University Hospital of Erlangen, Erlangen, Germany.
Wellner
Verena
V
Kämpgen
Eckhart
E
Schuler
Gerold
G
Schaft
Niels
N
D000951:Antigens, Neoplasm / Q000032:analysis* / Q000235:genetics; D002853:Chromatography, Liquid; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008543:Melanins / Q000032:analysis / Q000302:isolation & purification*; D008545:Melanoma / Q000737:chemistry* / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D000951:Antigens, Neoplasm; D008543:Melanins; D012334:RNA, Neoplasm