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Capture, amplification, and global profiling of microRNAs from low quantities of whole cell lysate.

ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression at the post-transcriptional level via a complex regulatory network that requires genome-wide miRNA profiling to dissect. The patterns of miRNA expression at the genome scale are rich in diagnostic and prognostic information for human diseases such as cancers. This analysis, however, requires multi-step purification of RNAs from large quantities of cells, which is not only time consuming and costly but also challenging in situations where cell numbers are limited. In this study, we report direct capture, amplification, and library preparation of miRNAs from whole cell lysate without the need of pre-purification. As a result, it enables genome-wide miRNA profiling reproducibly with low quantity of cell samples (?500 hematopoietic cells). Specifically, we conducted a systematic investigation of two key steps - cell lysis for miRNA release and 3' adaptor ligation required for direct miRNA capture and amplification. The obtained expression profile not only distinguishes cell types but also detects individual miRNA alterations in closely related isogenic cell lines. This approach, which is substantially simple as compared to the standard methods because of elimination of the need for RNA purification, is advantageous for the measurement of low quantity samples.

SUBMITTER: Wang N

PROVIDER: S-EPMC5605290 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Capture, amplification, and global profiling of microRNAs from low quantities of whole cell lysate.

Wang Nayi N Cheng Jijun J Fan Rong R Lu Jun J

The Analyst 20170801 17

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression at the post-transcriptional level via a complex regulatory network that requires genome-wide miRNA profiling to dissect. The patterns of miRNA expression at the genome scale are rich in diagnostic and prognostic information for human diseases such as cancers. This analysis, however, requires multi-step purification of RNAs from large quantities of cells, which is not only time consuming and costly but also challenging in s ...[more]

PMID: 28765841

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Project description:RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN's Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates.We found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P?<?0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison.Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.

Dataset Information

Capture, amplification, and global profiling of microRNAs from low quantities of whole cell lysate.

Publications

Capture, amplification, and global profiling of microRNAs from low quantities of whole cell lysate.

Similar Datasets

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