A high-throughput method to detect RNA profiling by integration of RT-MLPA with next generation sequencing technology.
RNA in formalin-fixed and paraffin-embedded (FFPE) tissues provides large amount of information indicating disease stages, histological tumor types and grades, as well as clinical outcomes. However, Detection of RNA expression levels in formalin-fixed and paraffin-embedded samples is extremely difficult due to poor RNA quality. Here we developed a high-throughput method, Reverse Transcription-Multiple Ligation-dependent Probe Sequencing (RT-MLPSeq), to determine expression levels of multiple transcripts in FFPE samples. By combining Reverse Transcription-Multiple Ligation-dependent Amplification method and next generation sequencing technology, RT-MLPSeq overcomes the limit of probe length in multiplex ligation-dependent probe amplification assay and thus could detect expression levels of transcripts without quantitative limitations. We proved that different RT-MLPSeq probes targeting on the same transcripts have highly consistent results and the starting RNA/cDNA input could be as little as 1 ng. RT-MLPSeq also presented consistent relative RNA levels of selected 13 genes with reverse transcription quantitative PCR. Finally, we demonstrated the application of the new RT-MLPSeq method by measuring the mRNA expression levels of 21 genes which can be used for accurate calculation of the breast cancer recurrence score - an index that has been widely used for managing breast cancer patients.
['Wang|Jing|J|', 'Yang|Xue|X|', 'Chen|Haofeng|H|', 'Wang|Xuewei|X|', 'Wang|Xiangyu|X|', 'Fang|Yi|Y|', 'Jia|Zhenyu|Z|', 'Gao|Jidong|J|']
D000328:Adult D000951:Antigens, Neoplasm D001943:Breast Neoplasms D005260:Female D020869:Gene Expression Profiling D059014:High-Throughput Nucleotide Sequencing D006801:Humans D008875:Middle Aged D009364:Neoplasm Recurrence, Local D016612:Paraffin Embedding D060888:Real-Time Polymerase Chain Reaction D015203:Reproducibility of Results
next generation sequencing RNA expression breast cancer 21-gene MLPA