Mol Biol Rep. 2016 Oct; 43(10):1165-78.

Identification of extracellular miRNA in archived serum samples by next-generation sequencing

Aarti Gautam1, Raina Kumar2, George Dimitrov2, Allison Hoke3, Rasha Hammamieh1 and Marti Jett4

1US Army Center for Environmental Health Research, 568 Doughten Drive, Fort Detrick, 21702-5010, MD, USA.

2Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research/Leidos-Biomedical Inc., Frederick, MD, 21702, USA.

3The Geneva Foundation, US Army Center for Environmental Health Research, Fort Detrick, MD, 21702, USA.

4US Army Center for Environmental Health Research, 568 Doughten Drive, Fort Detrick, 21702-5010, MD, USA.



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 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 the 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.

PMID: 27510798; DOI:10.1007/s11033-016-4043-6



microRNA (miRNA) are small, non-protein coding RNAs that are significant in gene regulation, are post-transcriptional regulators of gene expression, and are well conserved across species. The miRNAs found in tissues and body fluids, such as serum, remain stable for years even under severe conditions such as pH levels fluctuations, freeze-thaw cycles, and suboptimal storage conditions. Serum miRNAs, as well as other circulating miRNAs, are thought to influence the normal functionality of the circulatory and immune systems and may regulate every aspect of cellular activity. Serum miRNAs may be used for biomarker discovery in relation to health and disease status, and for studying the regulation of targeted gene expression. However, the technical hurdles encountered using serum miRNAs are low concentrations of miRNA and poor quantification.

The Department of Defense Serum Repository (DoDSR) contains serum samples that are augmented with the Defense Medical Surveillance System (DMSS), thus providing information about military and medical experiences and longitudinal data of the service members. In our pilot study, we utilized test samples from the DoDSR that were biologically compromised due to sub-optimal storage conditions after processing. The purpose of our study was to determine the feasibility of using comprised samples for down-stream processing for next-generation sequencing (NGS) technology.

To make systematic comparisons among the purification methods, the samples were processed in triplicate, either individually or as a homogenous pool, to help reduce the interpersonal differences among samples. A number of extraction kits have become available recently to optimize the extraction of small RNAs, and the extraction methods for this pilot study included different commercially available procedures. Several carriers, such as glycogen (Invitrogen, Life Technologies, Grand Island, NY, USA), bacteriophage MS2 (Roche, Basel, Switzerland) and yeast tRNA (Invitrogen) were used to maximize the yield of isolated RNA, and their effects in NGS were also investigated.

The quantification of miRNAs is below the limit of detection when using spectrophotometric devices and the use of the nucleic acid-based carriers made the accurate quantification of the samples impossible. The Agilent 2100 Bioanalyzer and equivalent instrumentation have the capability to obtain miRNA concentration using capillary electrophoresis, and this instrumentation was used to evaluate the presence of the RNA. Following initial extraction, the samples were prepared using the Illumina small RNA sample preparation procedure, followed by sequencing and mapping to the reference genome.

Our results illustrate that small RNAs have been preserved in the DoDSR archived serum samples and although each procedure tested was successful in extracting miRNA, some of the procedures performed better than others. Exosome precipitation prior to RNA extraction yielded a lower number of processed reads and did not produce greater amounts of miRNA as compared to the TRIzol-only procedure. The miRNeasy method produced similar numbers of processed reads from pooled and unpooled samples, and seemed to guarantee a constant amount of miRNA extracted across all corresponding samples. The addition of a carrier to TRIzol extraction caused inconsistency in the number of processed reads between pooled and unpooled samples, especially when bacteriophage MS2 or yeast tRNA carriers were added. Between pooled and unpooled samples, glycogen as a carrier had a more consistent number of reads and needs further investigation. Similar numbers of processed reads from pooled and unpooled samples were observed with the TRIzol-only extraction, but the extraction yielded low ratios of miRNA to small RNA. However, the use of the carrier during the TRIzol extraction increased the miRNA to small RNA ratio, with the exception of the use of yeast tRNA in the pooled samples.

To examine to size distribution of the small RNA, we assessed the read distribution plots and found that the addition of carrier to the extraction process increased the read length range. This effect could be attributed to degradation of the carrier, RNA degradation, or the occurrence of piwi-interacting RNA (piRNA), which is longer in length than miRNA. Upon aligning to miRBase, TRIzol extraction had the maximum percentage of aligned reads, whereas the miRNeasy extractions produced the minimum amount of aligned reads. While the addition of carriers produced the most processed reads, there was the lowest percentage of aligned sequences to miRBase. The use of carrier during the TRIzol procedure led to an increase in the proportion of miRNAs in unpooled samples, but did not result in a dramatic increase in the miRNA proportion in the pooled samples.

The method used to purify RNA impacts which miRNAs are isolated. Therefore, we examined the common miRNAs among the extraction procedures. In both the pooled and unpooled datasets, TRIzol extraction with MS2 carrier yielded the maximum miRNA overlap with the rest of the procedures. Since the pooled samples are derived from multiple samples, we expected an increase in the number of common miRNAs with the pooled samples as compared to the unpooled samples, which was evident in our data.

The purpose of this pilot study was to examine the feasibility of using the biologically compromised serum samples from the DoDSR for NGS, since the samples can be a valuable resource for studying diseases of military relevance. We were able to successfully extract total RNA in archived serum samples using all of the isolation methods, thus successfully sequencing the miRNAs from the archived serum. Our experiments indicated that TRIzol extraction with and without glycogen as a carrier and miRNeasy column extractions were the optimal methods for isolating the miRNAs from archived serum samples for best efficiency.


The contents of this article have been tailored from following journal article. 

Aarti Gautam, Raina Kumar, George Dimitrov, Allison Hoke, Rasha Hammamieh and Marti Jett. Identification of extracellular miRNA in archived serum samples by next-generation sequencing. Molecular Biology Reports 43, 1165-1178.



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