Small RNA & miRNA Sequencing by SOLiD Next-Generation Sequencing
Small noncoding RNAs are typically only ~18–40 nucleotides in length, but their effects on cellular processes are profound. Small RNAs have been shown to play critical roles in developmental timing, cell fate, tumor progression, and neurogenesis.
Animals, plants, and fungi contain several distinct classes of small RNAs, including:
- microRNA (miRNA)
- short interfering RNA (siRNA)
- piwi-interacting RNA (piRNA)
- repeat associated small interfering RNA (rasiRNA)
Small RNA Analysis With the 5500xl Genetic Analyzer
The SOLiD® Small RNA Solution is a robust method for hypothesis-neutral, whole genome analysis of expression patterns that enables you to discover novel RNA without the probe bias of microarrays. Using the SOLiD® Total RNA-Seq Kit, you can convert total RNA into a library in a single day. This simplified protocol reduces experimental variability and improves the detection of biologically relevant changes.
The SOLiD® Total RNA-Seq kit is a single kit for the construction of libraries either for small RNA or whole transcriptome analysis.
Together, the SOLiD® Total RNA-Seq Kit and the ultra–high-throughput 5500xl Genetic Analyzer:
- Conserve strand specificity of cDNA—allowing you to know exactly which strand a particular small RNA originated from
- Offer better sensitivity—high number of mapped reads coupled with the accuracy of exact call chemistry (ECC) give confidence in detecting rare and low-abundance small RNAs
- Offer versatile inputs—analyze small RNAs from a variety of sample types, including quality compromised samples, such as RNA from FFPE tissue, starting with as little as 5 ng Learn More
- Detect novel RNAs—facilitate detection of unmapped small RNAs and isoforms
- Allow multiplexing—sequence multiple small RNA libraries simultaneously in a single lane, helping to reduce the cost of analysis per sample
Step-by-Step Guide to Small RNA Analysis
Determine the level of sensitivity you need and the number of samples you want to analyze. Internal research conducted at Applied Biosystems suggests 10 million mappable sequences are needed for profiling known miRNAs in humans. Other organisms with similar sized genomes are expected to require a similar number of mappable sequences. It is not known at this time how many mappable sequences may be necessary to confidently discover new species of small RNAs.
User documentation for the 5500 Series Genetic Analysis Systems can be found on the SOLiD Community. Access is through the 5500 Series User Hub. Access is limited to 5500 Series Genetic Analysis Systems users who are registered on the solid community and have been authenticated through the process of requesting permission to join the 5500 Series User Hub.
The tools you need for each step in the Small RNA data analysis workflow:
|Data Analysis Step||Applied Biosystems Software||3rd-Party Software***|
|1. Align reads to reference in color space|
|2. Generate quality metrics||
|3. Generate sequencing and alignment statistics||
|4. Count and quanitate tags||
|5. Translate color space to base space|
|6. Visualize in context of annotation||
|7. Convert to SRF for publishing|
|*** Need XSQ --> csfasta/qual converter|
- miRNA Discovery and Profiling Using the SOLiD® Small RNA Expression Kit
- Whole Genome Analysis of Small RNA
Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors
Publication: PLoS One
A Comprehensive Survey of miRNA Repertoire and 39 Addition Events in the Placentas of Patients with Pre- Eclampsia from High-Throughput Sequencing
Publication: PloS One, (2011)
Authors: Guo. et al.
Next-Generation Sequencing of MicroRNAs for Breast Cancer Detection
Publication: Journal of Biomedicine and Biotechnology (2011)
Authors: Qu Q. et al.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.