High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. Export

@article{citeulike:3471732, abstract = {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.}, author = {Mestdagh, P. and Feys, T. and Bernard, N. and Guenther, S. and Chen, C. and Speleman, F. and Vandesompele, J.}, citeulike-article-id = {3471732}, citeulike-linkout-0 = {http://dx.doi.org/10.1093/nar/gkn725}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/18940866}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=18940866}, day = {21}, doi = {10.1093/nar/gkn725}, issn = {1362-4962}, journal = {Nucleic acids research}, keywords = {mirna, rt-pcr, technology}, month = {December}, number = {21}, posted-at = {2009-11-09 14:45:52}, priority = {1}, title = {High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA.}, url = {http://dx.doi.org/10.1093/nar/gkn725}, volume = {36}, year = {2008} }

TY - JOUR ID - citeulike:3471732 L3 - citeulike-article-id:3471732 N2 - 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. IS - 21 JF - Nucleic acids research SN - 1362-4962 TI - High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. VL - 36 KW - mirna KW - rt-pcr KW - technology AU - Mestdagh, P AU - Feys, T AU - Bernard, N AU - Guenther, S AU - Chen, C AU - Speleman, F AU - Vandesompele, J PY - 2008/12/21/ UR - http://dx.doi.org/10.1093/nar/gkn725 ER -