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RNA-Seq: A Comprehensive Alternative to Microarray for Transcriptome Analysis

RNA-Seq is an advanced method for analyzing genome-wide gene expression. It involves growing cells or isolating specific tissues like brain, liver, or muscle, followed by total RNA extraction and mRNA isolation using poly(A)+ selection. The extracted RNA is then reverse-transcribed to cDNA, which undergoes sequencing. This process includes steps like cluster amplification and sequencing via Solexa technology, providing an in-depth analysis of known genes and uncharacterized regions. RNA-Seq enables researchers to assess differential gene expression and understand transcriptomic variations with greater sensitivity than traditional methods.

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RNA-Seq: A Comprehensive Alternative to Microarray for Transcriptome Analysis

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  1. RNA-Seq An alternative to microarray

  2. Grow cells or isolate tissue (brain, liver, muscle) Isolate total RNA Isolate mRNA from total RNA (poly A+ select) Fragment RNA Make and amplify cDNA Sequence the ends of the cDNA Map the sequences to the human genome Count the number of sequence tags at each known gene (and at locations for which no gene is known) Correct for background Analyze the data Steps

  3. PROCEDURE - Solexa –Cluster Amplification 1) Load Samples to Flow Cell 2. Attach DNA to Surface 3. Bridge Amplification 8 Lanes are loaded onto the flow cell for simultaneous analysis Single stranded DNA fragments bind randomly to the inside surface of the flow cell. Unlabeled nucleotides and enzyme are added to initiate solid-phase bridge amplification.

  4. PROCEDURE - Solexa –Cluster Amplification 4. Fragments Become Double Stranded 5. Double Stranded Molecules are Denatured 6. Amplification is Completed The enzyme incorporates nucleotides to build double stranded bridges on the solid-phase substrate. Denaturation leaves single-stranded template anchored to the substrate Several million dense clusters of double stranded DNA are generated in each channel of the flow cell.

  5. PROCEDURE- Solexa Sequencing & Genome Analyzer 1. Determine 1st Base 2. Image 1st Base 3. Determine 2nd Base The first sequencing cycle is initiated by adding all 4 labeled reversible terminators, primers, and DNA polymerase to the flow cell After laser excitation, an image of the emitted fluorescence from each cluster on the flow cell is captured. The 2nd sequencing cycle is initiated by adding all 4 labeled reversible terminators and enzymes.

  6. PROCEDURE- Solexa Sequencing & Genome Analyzer 4. Image 2nd Base 5. Sequence Read Continues Over Multiple Chemistry Cycles 6. Align and Map Data After laser excitation, image data is collected like before. The identity of the 2nd base for each cluster is recorded. 35 cycles of sequencing are repeated to determine the sequence of bases in a given fragment a single base at a time. Align data and map the sequences to the reference genome.

  7. What can you learn with each one? What can you learn from one but not from the other? How is the primary data acquired? How are systematic biases eliminated? How do you normalize How would you look for differential expression? How would you cluster? How can you combine data from multiple experiments? Which is more sensitive? What kinds of additional software do you need? What are the similarities and differences?

  8. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods (2008) 5: 621. http://www.illumina.com/pages.ilmn?ID=203 References

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