The DOI matches its Crossref record; 20 of the 20 references it cites were found in a registry or an index.
High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA
Open accessAbstract
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.
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Cites
MicroRNA expression profiles classify human cancers
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Oncomirs—MicroRNAs with a role in cancer
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Real-time quantification of microRNAs by stem-loop RT-PCR
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Self-citationc-Myc-regulated microRNAs modulate E2F1 expression
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An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues
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A molecular programme for the specification of germ cell fate in mice
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A custom microarray platform for analysis of microRNA gene expression
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Microarray-based, high-throughput gene expression profiling of microRNAs
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“Briefly, 8ml of total RNA (50 ng/ml) was supplemented with RT primer mix ( 10 ), RT buffer (10), MultiScribe Reverse Transcriptase (10 U/ml), dNTPs with dTTP (0.5 mM each), MgCl2 (3 mM) and AB RNase inhibitor (0.25 U/ml) in a total reaction volume of 80ml.”
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A novel and universal method for microRNA RT-qPCR data normalization
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Self-citationKim, A. H., Reimers, M., Maher, B., Williamson, V., McMichael, O., McClay, J. L., van den Oord, E. J. C. G., Riley, B. P., Kendler, K. S., & Vladimirov, V. I. (2010) · Schizophrenia Research · doi:10.1016/j.schres.2010.07.002 · Cited by 298
A novel mean-centering method for normalizing microRNA expression from high-throughput RT-qPCR data
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Metadata
- Authors
- Mestdagh, P., Feys, T., Bernard, N., Guenther, S., Chen, C., Speleman, F., & Vandesompele, J.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2008-10-21
- Volume
- 36
- Issue
- 21
- Pages
- e143-e143
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2140741841
- Cited by
- 288
- References
- 20
- FWCI
- 8.56
- Type
- journal article
- Language
- en
- Open access
- gold
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