Abstract
miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-016-4043-6) contains supplementary material, which is available to authorized users.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.miRNA is a functionally important class of small, non– protein coding RNA, 19–24 nucleotides in length, that are post-transcriptional regulators of gene expression [1]. miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation [2]
Mol Biol Rep (2016) 43:1165–1178 indicate that more than one-third of the cellular transcriptome is regulated by miRNAs [7]. miRNAs have been found in tissues, serum/plasma, and other body fluids in a stable form that is protected from endogenous RNase activity. miRNA expression in biofluids, such as serum, plasma, urine or cerebrospinal fluid, is a rapidly expanding area of research [8]. miRNAs in circulation are thought to contribute to the normal functioning of circulatory and immune systems [9, 10] and have been proposed as candidate biomarkers of health/disease status [11, 12]
The purpose of our study was to develop a method of RNA extraction from archived serum samples stored at -30 °C to obtain sufficient yields and acceptable quantities of miRNA for NextGeneration Sequencing (NGS). miRNA has been shown to be present in twelve body fluids tested [36], and higher miRNA concentrations have been observed in serum samples as compared to plasma samples [37]
Summary
Electronic supplementary material The online version of this article (doi:10.1007/s11033-016-4043-6) contains supplementary material, which is available to authorized users.miRNA is a functionally important class of small, non– protein coding RNA, 19–24 nucleotides in length, that are post-transcriptional regulators of gene expression [1]. miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation [2]. MiRNA is a functionally important class of small, non– protein coding RNA, 19–24 nucleotides in length, that are post-transcriptional regulators of gene expression [1]. MiRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation [2]. MiRNAs have been found in tissues, serum/plasma, and other body fluids in a stable form that is protected from endogenous RNase activity. MiRNA expression in biofluids, such as serum, plasma, urine or cerebrospinal fluid, is a rapidly expanding area of research [8]. The use of serum or plasma reduces variables and possibly allows the detection of a more concentrated disease-related miRNome by decreasing the background expression of miRNAs from blood cells
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