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Gene Expression - RNA-seq etc, Gene Expression - Coggle Diagram

- - - - take mRNA from cell and reverse transcribe to complementary DNA (cDNA), then tag with fluorescence
      - seed a microarray (with cells) with ssDNA from different genes of interest. (Many copies of ssDNA by PCR)
        
        Hybridise fluorescent cDNA (flow through the cells). Wherever gene expression is high, that particular spot/cell in the microarray will give out high intensity fluorescence
        
        This is hence measuring expression of multiple genes for a given condition and time.
    - - Use of WGS
        
        Extract mRNA from all RNA
        
        Remove contaminants and fragment randomly
        
        Reverse transcribe to cDNA
        
        Ligate with adaptors and do PCR amplification
        
        Sequence the fragments
        
        2 more items...
        
        Minimize PCR amplification to avoid amplification bias, which can interfere with the relative proportions of the fragments and hence genes
        
        Most RNA in cells is rRNA (>87%), tRNA and other non-coding ncRNA (>95%)
        
        Coding mRNA is 3' polyadenylated with introns spliced out in eukaryotes-- it is a natural molecular tag -- hence is used to enrich sample with mRNA
        
        RNA-seq zoom lecture, slide 11
        
        Better way if you want to extract mature mRNA from RNA sample
        
        Another option is rRNA reduction - Addition of rRNA specific oligos and subsequent degradation by RNAseH
      - Other uses - Detection of SNPs, splice variants, gene fusions
      - DGE Analysis steps - lecture 14, slide 20
      - miRNA-seq
        
        MicroRNAs (miRNAs) are regulatory RNAs that are expressed in many cells but do not code for proteins.
        
        Belong in intron regions - sometimes at splice junctions
        
        Assignment 4 word answers last page
        
        RNA-seq protocols to examine the expression differences in miRNAs when investigating basic cell function and disease processes.
        
        They can be changed based on developmental stage, drug treatment and disease.
        
        The expression levels of miRNAs are regulated by cells.
        
        Read length - 50 bp, single end sequencing (reads would be longer than miRNA sequences)
        
        Adaptors often remain in sequences due to their short lengths. Hence, adaptor trimming is necessary to improve read alignments
        
        Challenges
        
        Small read lengths -- multi mapping to repeat regions
        
        RNA editing can cause differences between the mature miRNA sequence and the genomic sequence, leading to additional alignment challenges.
        
        Any small error (like a SNP or sequencing errors) can have a big impact on alignment due to smaller size of read.
      - Considerations for design of analysis
        
        guest lect. rna-seq - slide 6
  - - - Input lots of RNA, gel electrophoresis, capture RNA of interest using ssDNA probes
      - only one gene at a time
    - - Reverse transcribe RNA, tag flourescence and amplify with PCR
        
        If the required fluorescence is detected with just fewer cycles of PCR, then high concentration of template (original RNA) was already present in the sample. Else, the gene expression is lower.
      - Few genes at a time