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Overview of SOLiD® Total RNA-Seq Kit Workflow Click to enlarge.

Global transcriptome analysis is rapidly growing in prominence, providing insight into research on complex conditions including cancer, diabetes, and heart disease.

The accuracy and throughput of the 5500xl Genetic Analyzer enables unparalleled analysis of RNA expression, or RNA-Seq.

Advantages Over Microarrays
Because microarrays are hybridization-based, they cannot detect RNA transcripts that are not included in your array design or from repeated sequences. In addition, microarrays offer limited dynamic range to detect subtle changes in expression levels, a critical component of understanding biological responses to environmental stimuli.

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Strand-Specific Read Distribution
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Detect All Known and Novel RNAs in a Transcriptome

The sequencing-based SOLiD® Total RNA-Seq Kit enables you to detect all known and novel RNAs present in biological samples, with no bias toward known RNA molecules (a common drawback with probe-based technologies). RNA-Seq provides you with new views of a cell’s transcriptome, including:

  • Expression of all coding and noncoding RNAs
  • Identification of alternative splicing events
  • Expressed single nucleotide polymorphisms (SNPs) or mutations
  • Translocations and fusion transcripts
  • Allele-specific expression patterns

Together, the SOLiD® Total RNA-Seq Kit and the Ultra–High-Throughput 5500xl Genetic Analyzer Allow You to:

  • Conserve strand specificity of cDNA—discern between overlapping RNAs transcribed from the sense or antisense strand
  • Increase read number—generate greater than 700 million mapped sequence reads per FlowChip for RNA expression analysis
  • Quantify critical parameters—ERCC Spike-In Control Mixes allow you to calculate changes in expression level, determine lower limits of detection, and calculate your dynamic range with a high degree of confidence.
  • Detect novel RNAs—detect fusion transcripts and alternate splicing with reverse sequencing on the same read
  • Multiplex—sequence up to 96 RNA libraries simultaneously; reduce the cost of analysis per sample.

Step-by-Step Guide to Whole Transcriptome Analysis Products

Determine the level of sensitivity you need and the number of samples you want to analyze. The number of mappable sequences you need is dependent on the:

  • Genome size of the organism you are studying
  • Complexity of the RNA fraction you are studying

For instance, for the human genome and other similar sized genomes, the current literature suggests you need 50 million mappable sequences to identify known RefSeqs. It is not known how many mappable sequences are needed to discover all relevant RNAs and different alleles of RNAs.

Isolate RNA from various sample types, including blood, cultured cells, and tissues.

Technical Considerations:

  • High-quality RNA is critical for library generation

The tools you need for each step in the Whole Transcriptome data analysis workflow:

Data Analysis Step Applied Biosystems Tools Applied Biosystems Software 3rd-Party Software
1. Align transcriptome reads to a genome  
2. Generate alignment statistics    
3. Count tags for genes or exons  
4. Export data in base space    
5. Visualize data    
6. Convert to SRF for publishing    

Application Notes