Lecture 2 - Transcriptomic I (Transcriptome, DNA/RNA, Microarrays)

What is a transcriptome?

• study of an organism’s enitre set of transcripts encoded within the genome (mainly mRNA + some other types)

• Includes all transcripts produced within an organism, organ, tissue, or cell type

• transcripts deposited at right place and right time

What are some examples of RNA?

• mRNA - messenger RNA

• tRNA - transfer RNA

• rRNA - ribosomal RNA

• miRNA - microRNA

• siRNA

• small RNA

• piRNA - PIWI RNA

• pre-mRNA - unprocessed mRNA

What kind of RNA does most of transcriptomics characterize?

• mRNA

• emphasis on Central Dogma - creating mRNA and converting it to a protein

What is the correlation between transcripts and protein abundance? What may cause slight deviances?

• Pearson correlation of 0.7

  • strong correlation

• not all mRNA converted due to:

  • gene silencing

  • post-trxn modifications

  • mRNA degradation

How is DNA synthesized and in what direction?

• DNA synthesis requires:

  • nucleotides or dNTPs

  • DNA polymerase

  • Primer

• Goes in 5’ to 3’ direction

Purine Nucleotides (one ring)

• Adenine and Guanine

• two hydrogen bonds

Pyrimidine nucleotides (two rings)

• Cytosine and Thymine and Uracil

• three hydrogen bonds

Similarities and Differences between RNA and DNA

Similarities:

• Both contain A, C, G

Differences:

• RNA has 2’ OH while DNA does not

• DNA has thymine (T), RNA has Uracil (U)

• RNA mainly single stranded (but can be double stranded), DNA mainly double stranded

Which DNA strand does the mRNA represent/match?

It matches the coding/non-template strand, NOT the noncoding/template strand

What are 3 processes involved in Cotranscriptional processing of mRNA?

1) 5’ methyl guanosine cap

2) splicing of intron, joining of exons

3) poly A tail (help export and protect mRNA)

All of these processes occur around the same time, also involves a holoenzyme moving 5’ to 3’

What things are involved in the synthesis of cDNA (only exons compared to regular DNA) from RNA?

• oligodt primner (TTTT and 5’)

  • connects to the poly A tail on 3’ end

• synthesis occurs from 5’ → 3”

• addition of dNTPs (building blocks)

• enzyme - reverse transcriptase

cDNA - DNA synthesized from processed mRNA

What is the purpose of a microarray?

It is a tool used to measure gene expression and detect DNA sequence varaition by looking at relative concentration of nucleic acid in a mixture

What three things are required for a microarray?

• substrate - glass, plastic, or nylon membrane (chip)

• probe - ssDNA printed of synthesized on substrate, arranged in grid-like manner; cDNA or oligonucleotide

• target - labelled ssDNA generated from a biological sample used to query the probe

Microarray probes

• Involve oligonucleotides 20-30 bp in length

• 10-20 oligo nucleotides per gene (some perfect, some mismatched)

• Oligonucleotides are selected to be:

  • unique in the genome

  • non-overlapping

Target Labeling

• cDNA made using reverse transcriptase

• Fluorescently labeled nucleotides added

• Labled nucleotides incorporated into cDNA

Scanning of microarrays

• Laser beam excites each spot of DNA

• Takes amount of fluorescence detected

• Take average of light intensity, take ratio of array to average

• higher intensity = higher transcripts = higher expression of gene

Hybridization

• Results given as ratios

• Images use colors (blue = absent, white = high)

What are the disadvantages of Microarrays?

Cons

• Requires a sequenced genome (non genome = no chip)

• Only works well on studied/know organisms, not for novel/newly observed organisms

• Often not comprehensive of entire genome

• Uses relative fluorescence intensity, so exact intensity may not be known

• Depends on hybridization for data/results to be evident

What are the advantages of Microarrays?

Pros

• Can use a small amount of amplified material

• Well-established normalization algorithms

• Easy to use