Small RNA Analysis
Small non-coding RNAs are typically only ~18–40 nucleotides in length, however their effect on cellular processes is profound (see Figure 1). Small RNA has been shown to play critical roles in developmental timing, cell fate, tumor progression, neurogenesis.
Animals, plants, and fungi contain several distinct classes of small RNA, including:
- microRNA (miRNA)
- short interfering RNA (siRNA)
- piwi-interacting RNA (piRNA)
- siRNA RNA (rasiRNA)
Small RNA Analysis with the SOLiD™ System
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 suitable for emulsion PCR 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 for either small RNA or whole transcriptome analysis. The expression profiles from these libraries are highly concordant with those from previous kits (SOLiD™ RNA Expression and SOLiD™ Whole Transcriptome Analysis kits).
Together, the SOLiD™ Total RNA-Seq Kit and the ultra-high throughput SOLiD™ 4 System:
- Conserve strandedness of cDNA, allowing you to know exactly which strand a particular transcript originated from.
- Generate up to greater than 700 million mappable sequence reads per run for a fragment library enabling high sensitivity for analyzing low RNA expression levels and measuring accurate fold changes at the same low expression levels.
- Enable you to multiplex and sequence RNA libraries simultaneously, reducing the cost of analysis per sample.
- Detect allele specific expression patterns
Experimental Workflow: Find the Products You Need for Every Step
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Step 1: Design Experiment
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.
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Step 2: Isolate RNA
Isolate RNA from various sample types, including blood, cultured cells, and tissues.
Technical Considerations:
- High-quality RNA is critical for library generation
Products for Step 2: Isolate RNA
For isolating total RNA from cells and tissues:
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mirVana™ miRNA Isolation Kit
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Isolate total RNA that contains small RNAs such as miRNA, siRNA, and snRNA. These small RNAs are often lost during traditional RNA isolation methods.
For isolating small RNA from cells and tissues:
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View more reagents, kits and bench-top devices on invitrogen.com
PureLink™ miRNA Isolation Kit
The PureLink™ miRNA Isolation Kit is uniquely designed to purify cellular small (< 200 nt) RNA molecules, including tRNA, 5S rRNA, and 5.8S rRNA, and regulatory RNA molecules such as microRNA (miRNA) and short interfering RNA (siRNA). Purified RNA is suitable for northern blot analysis and microarray analysis.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Step 3: Construct cDNA Library
Generate cDNA by reverse transcription from adaptors ligated to ends of the small RNA molecule. Amplify the cDNA using primers complementary to adaptors, and then purify it.
Products for Step 3: Construct cDNA Library
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SOLiD™ Total RNA-Seq Kit
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A single kit for the rapid construction of libraries for either whole transcriptome discovery or small non-coding RNA analysis.
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SOLiD™ RNA Barcoding Kit
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Compatible with the SOLiD™ Total RNA-Seq Kit, the SOLiD™ RNA Barcode Kits contain barcodes with unique sequences designed for optimal multiplexing.
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Step 4: Amplify & Sequence
After constructing cDNA libraries, amplify them using standard emulsion PCR. After amplification, deposit the beads on a slide for sequencing on the SOLiD™ Analyzer.
Products for Step 4: Amplify & Sequence
Sequencing
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SOLiD™ 4 System
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The Applied Biosystems SOLiD™ 4 System is a revolutionary genetic analysis platform that enables massively parallel sequencing of clonally-amplified DNA fragments linked to beads. The sequencing methodology is based on sequential ligation with dye-labeled oligonucleotides. The SOLiD™ 4 System enables researchers to obtain higher-quality genomes at lower cost without the purchase of a new instrument.
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Fragment Library Sequencing Reagents for SOLiD™ 4 System, Main Kits
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These kits contain sequencing master mixes in an easy-to-use strip tube format for ligation sequencing of fragment libraries on the SOLiD™ 4 System. Reagent mixing is not required, minimizing tube mix ups and providing a convenient automated method for sequencing reagent handling.
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Instrument Buffers for SOLiD™ 4 System
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This kit contains a set of buffers needed for ligation sequencing on the SOLiD™ 4 System and is sufficient for 110 cycles. The product is compatible only with the SOLiD™ 4 System. Store components at ambient, 2 °C to 8 °C, and at -15 °C to -25 °C.
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Workflow Analysis Reagents for SOLID™ 4 System
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This kit is for accurate qualification and quantitation of templated beads prior to your sequencing run. Use this kit to perform a workflow analysis (WFA) run on the SOLiD™ 4 Analyzer.
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Barcoding Sequencing Reagents for SOLiD™ 3 Plus
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Emulsion PCR (ePCR)
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SOLiD™ ePCR Kits
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The SOLiD™ ePCR Kits contain the components needed for amplifying short library inserts by emulsion PCR on beads. Each kit includes reagents for performing PCR, reagents for generating emulsions, and P1 DNA beads for clonal amplification.
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SOLiD™ Buffer Kits
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These kits contain buffers needed for emulsion PCR, bead enrichment, and bead deposition onto slides for ligation sequencing. These kits are available a 10 full-scale reaction size and a 20 mini-reaction size.
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Thermocycler Systems
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We have a full range of thermal cyclers for every budget, including our award-winning Veriti® thermal cycler series.
Bead Deposition
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SOLiD™ Bead Deposition Kits
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These kits contains components for depositing templated beads onto a slide for ligation sequencing on the SOLiD™ Analyzer. The kits contain reagents for performing a 3’ end modification and buffers. These kits are available in a 10 full-scale reaction size and a 20 mini-reaction size.
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SOLiD™ Bead Enrichment Kits
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These kits contain components to enrich templated beads amplified by emulsion PCR. The kit includes enrichment beads and reagents. These kits are available in a 10 full-scale reaction size and a 20 mini-reaction size.
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SOLiD™ Slide Kits
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These kits contain activated slides and storage buffer for ligation sequencing on the SOLiD™ Analyzer.
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Step 5: Analyze Data
Figure 1
Using the data analysis tools listed below:
- Align reads to databases of known small RNAs
- Align reads to a reference genome
- Translate color space to base space
- Visualize annotated data
The SOLiD™ System provides a highly reproducible method for gene expression analysis (Figure 1) with a dynamic range of 5-6 logs.
Products for Step 5: Analyze Data
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Data Analysis Tools
The tools you need for each step in small RNA data analysis workflow: | Data Analysis Step | Applied Biosystems Tools | Applied Biosystems Software | 3rd-Party Software |
| 1. Align tags reads to reference in color space. | | | |
| 2. Generate sequencing and alignment statistics. | | | |
| 3. Count and quantitate tags. | | | |
| 4. Translate color space to base space. | | | |
| 5. Visualize in context of annotation. | | | |
| 6. Convert to SRF for publishing. | | | |
Step 6: Validate Results
SOLiD™ System and TaqMan data
compared across 244 miRNA.
Validate your SOLiD™ System results with TaqMan® MicroRNA Assays.
TaqMan® MicroRNA Assays are:
- Gold standard validation of SOLiD™ System results
- Highly specific - quantitate only the biologically active
- mature miRNAs Fast, simple and scalable
Products for Step 6: Validate Results
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TaqMan® MicroRNA Assays
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TaqMan® MicroRNA Assays quantitate microRNAs with the specificity and sensitivity of TaqMan Assay chemistry. A simple, two-step protocol requires only reverse transcription with a miRNA-specific primer, followed by real-time PCR with TaqMan probes.
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Custom TaqMan® Small RNA Assays
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Applied Biosystems pioneered the use of TaqMan® Real-Time PCR Assays to accurately and specifically measure the expression of mature microRNA (miRNA). Now this proven technology is available for the analysis of any small RNA molecule with Custom TaqMan® Small RNA Assays. The novel adaptations in TaqMan® Assay design developed for the study of miRNAs are ideal for analysis of any small nucleic acid less than 200 bases long, including newly discovered miRNAs, Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA).
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Real-Time PCR Instruments
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Application Notes
Poster
Publication
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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