MicroRNA sequencing (miRNA-seq), a type of RNA-Seq, is the use of next-generation sequencing or massively parallel high-throughput DNA sequencing to sequence microRNAs, also called miRNAs. miRNA-seq differs from other forms of RNA-seq in that input material is often enriched for small RNAs. miRNA-seq allows researchers to examine tissue-specific expression patterns, disease associations, and isoforms of miRNAs, and to discover previously uncharacterized miRNAs. Evidence that dysregulated miRNAs play a role in diseases such as cancer has positioned miRNA-seq to potentially become an important tool in the future for diagnostics and prognostics as costs continue to decrease. Like other miRNA profiling technologies, miRNA-Seq has both advantages (sequence-independence, coverage) and disadvantages (high cost, infrastructure requirements, run length, and potential artifacts).
Introduction MicroRNAs (miRNAs) are a family of small ribonucleic acids, 21-25 nucleotides in length, that modulate protein expression through transcript degradation, inhibition of translation, or sequestering transcripts. The first miRNA to be discovered, lin-4, was found in a genetic mutagenesis screen to identify molecular elements controlling post-embryonic development of the nematode Caenorhabditis elegans. The lin-4 gene encoded a 22 nucleotide RNA with conserved complementary binding sites in the 3'-untranslated region of the lin-14 mRNA transcript and downregulated LIN-14 protein expression. miRNAs are now thought to be involved in the regulation of many developmental and biological processes, including haematopoiesis (miR-181 in Mus musculus), lipid metabolism (miR-14 in Drosophila melanogaster) and neuronal development (lsy-6 in Caenorhabditis elegans). These discoveries necessitated development of techniques able to identify and characterize miRNAs, such as miRNA-seq.
History MicroRNA sequencing (miRNA-seq) was developed to take advantage of next-generation sequencing or massively parallel high-throughput sequencing technologies in order to find novel miRNAs and their expression profiles in a given sample. miRNA sequencing in and of itself is not a new idea, initial methods of sequencing utilized Sanger sequencing methods. Sequencing preparation involved creating libraries by cloning of DNA reverse transcribed from endogenous small RNAs of 21–25 bp size selected by column and gel electrophoresis. However, this method is exhaustive in terms of time and resources, as each clone has to be individually amplified and prepared for sequencing. This method also inadvertently favors miRNAs that are highly expressed. Next-generation sequencing eliminates the need for sequence specific hybridization probes required in DNA microarray analysis as well as laborious cloning methods required in the Sanger sequencing method. Additionally, next-generation sequencing platforms in the miRNA-SEQ method facilitate the sequencing of large pools of small RNAs in a single sequencing run. miRNA-seq can be performed using a variety of sequencing platforms. The first analysis of small RNAs using miRNA-seq methods examined approximately 1.4 million small RNAs from the model plant Arabidopsis thaliana using Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS) sequencing platform. This study demonstrated the potential of novel, high-throughput sequencing technologies for the study of small RNAs, and it showed that genomes generate large numbers of small RNAs with plants as particularly rich sources of small RNAs. Later studies used other sequencing technologies, such as a study in C. elegans which identified 18 novel miRNA genes as well as a new class of nematode small RNAs termed 21U-RNAs. Another study comparing small RNA profiles of human cervical tumours and normal tissue, utilized the Illumina (company) Genome Analyzer to identify 64 novel human miRNA genes as well as 67 differentially expressed miRNAs. Applied Biosystems SOLiD sequencing platform has also been used to examine the prognostic value of miRNAs in detecting human breast cancer.
Methods
Small RNA Preparation
Sequence library construction can be performed using a variety of different kits depending on the high-throughput sequencing platform being employed. However, there are several common steps for small RNA sequencing preparation. Total RNA Isolation In a given sample all the RNA is extracted and isolated using an isothiocyanate/phenol/chloroform (GITC/phenol) method or a commercial product such as Trizol (Invitrogen) reagent. A starting quantity of 50-100 μg total RNA, 1 g of tissue typically yields 1 mg of total RNA, is usually required for gel purification and size selection. Quality control of the RNA is also measured, for example running an RNA chip on Caliper LabChipGX (Caliper Life Sciences). Size Fractionation of small RNAs by Gel Electrophoresis Isolated RNA is run on a denaturing polyacrylamide gel. An imaging method such as radioactive 5'-32P-labeled oligonucleotides along with a size ladder is used to identify a section of the gel containing RNA of the appropriate size, reducing the amount of material ultimately sequenced. This step does not have to be necessarily carried out before the ligation and reverse transcription steps outlined below. Ligation The ligation step adds DNA adaptors to both ends of the small RNAs, which act as primer binding sites during reverse transcription and PCR amplification. An adenylated single strand DNA 3'adaptor followed by a 5'adaptor is ligated to the small RNAs using a ligating enzyme such as T4 RNA ligase2. The adaptors are also designed to capture small RNAs with a 5' phosphate group, characteristic microRNAs, rather than RNA degradation products with a 5' hydroxyl group. Reverse Transcription and PCR Amplification This step converts the small adaptor ligated RNAs into cDNA clones used in the sequencing reaction. There are many commercial kits available that will carry out this step using some form of reverse transcriptase. PCR is then carried out to amplify the pool of cDNA sequences. Primers designed with unique nucleotide tags can also be used in this step to create ID tags in pooled library multiplex sequencing.
Sequencing
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