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Updated: Apr 17, 2026
Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
Published on: November 18, 2014
Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total
Savita Shanker1, Ariel Paulson1, Howard J Edenberg1
11 Genomic Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32610, USA; 2 Stowers Institute for Medical Research, Kansas City, Missouri 64112, USA; 3 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA; 4 Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA; 5 Research Technology Support Facility, Office of the Vice President for Research and Graduate Studies, Michigan State University, East Lansing, Michigan 48824, USA; 6 DNA Core Facility,Office of Research, University of Missouri, Columbia, Missouri 65211, USA; 7 New Jersey Medical School-Molecular Resource Facility, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, New Jersey 08863, USA; 8 College of Medicine, Laboratory for Molecular Biology and Cytometry Research, Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA; 9 Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA; 10 Department of Orthopaedic Surgery and Biomedical Engineering, College of Medicine, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA; 11 Genomics Core Facility Center for Genetic Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA; 12 Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth College, Hanover, New Hampshire 03756, USA; 13 University of Michigan DNA Sequencing Core, Biomedical Research Core Facilities, University of Michigan, Ann Arbor, Michigan 48109, USA; 14 Department of Biochemistry and Molecular Medicine, University of California-Davis, School of Medicine, Sacramento, California 95817, USA; and 15 Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los
Abstract:
This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.
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