Comparison of RNA amplification techniques meeting the demands for the expression profiling of clinical cancer samples.
Available ribonucleic acid (RNA) amplification methods are extensively tested for reproducibility, but only a few studies additionally deal with potential amplification bias. On targeted arrays, we evaluated three amplification protocols, which are less time consuming than the commonly used T7-RNA polymerase based in vitro transcription protocols and therefore may be more suitable for clinical use: Template-switching polymerase chain reaction (PCR), Ribo-single primer isothermal amplification and a random primer-based PCR. Additionally, a more sensitive labelling method, Dendrimer labelling, was evaluated. All methods were compared to unamplified RNA labelled at reverse transcription. From our results, we conclude that RNA amplification with template-switching PCR is highly reproducible and results in a reliable representation of the starting RNA population. We then assessed whether RNA amplification of clinical breast and thyroid cancer samples with template-switching PCR showed robust performance when altered cycle numbers or partially degraded RNA were used. Template-switching PCR proved to be a very reliable method for global RNA amplification, even when starting from partially degraded RNA down to a RNA Integrity Number of 4.3. In conclusion, template-switching PCR amplification promises to help micro-array expression profiling of limited amounts of human samples on its way to a clinical routine.
Lauss
Martin
M
Austrian Research Centers GmbH-ARC, 2444 Seibersdorf, Austria. martin.lauss@arcs.ac.at
Vierlinger
Klemens
K
Weinhaeusel
Andreas
A
Szameit
Sandra
S
Kaserer
Klaus
K
Noehammer
Christa
C
D001435:Bacteriophages; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics* / Q000473:pathology; D002291:Carcinoma, Papillary / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D013696:Temperature; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology; D014764:Viral Proteins / Q000235:genetics
D012334:RNA, Neoplasm; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases