High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA
| Autor: | Caifu Chen, Jo Vandesompele, Nathalie Bernard, Pieter Mestdagh, Simone Guenther, Franki Speleman, Tom Feys |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Regulation of gene expression
Oncogene Proteins N-Myc Proto-Oncogene Protein Reverse Transcriptase Polymerase Chain Reaction Gene Expression Profiling RNA Nuclear Proteins Computational biology Biology Stem-loop Molecular biology Reverse transcriptase Gene expression profiling MicroRNAs Real-time polymerase chain reaction Gene Expression Regulation Cell Line Tumor microRNA Gene expression Genetics Methods Online Humans DNA Primers |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods. Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step. The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling. |
| Databáze: | OpenAIRE |
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