Week 6 Transcriptomics

functional genomics

global approach to the study, function, expression, and interaction of gene products

transcriptome

sequence and expression patterns of all RNA transcripts (where, when, how much)

proteome

sequence and expression patterns of all proteins (where, when, how much)

interactome

complete set of physical interactions between proteins and DNA sequence, between proteins an RNAs, and between proteins

2 components of transcriptome

1. mRNA

2. ncRNA

mRNA

messanger RNA: translated into proteins, and often less then 5% of the total RNA content of a cell

what are ncRNA and what are the 4 main types

ncRNA: not translated into protein but have important functions in the cell

1. rRNA

2. tRNA

3. sncRNA

4. lncRNA

how many RNA pol in eukaryotes and what do they transcribe

RNA pol1: transcribes rRNA genes

RNA pol 2: transcribes mRNA genes

RNA pol 3: transcribes tRNA genes

what are the 2 kinds of nuclear sncRNA’s and when were they discovered

discovered in the 1960’s

1. small nuclear RNAs (snRNA)

2. small nucleolar RNAs (snoRNA)

snRNA

• most are found in snRNPs in spliceosome

• example. U-1snRNA, U2-snRNA, range from 105-190 nucleotides

• U7-snRNA functions in processing of histones mRNAs

snoRNA

• nucleolar localization

• chemical modifications of rRNAs (methylation to pseudouridylation)

• can also modify snRNAs in Cajal bodies (scaRNA’s- small Cajal specific RNAs)

2 types of cytoplasmic sncRNAs and when were they discovered

discovered in the 1990’s

1. short interfering RNAs (siRNAs)

2. microRNAs (miRNAs)

siRNA

• 20-25 nucleotide in length

• involved in RNA interference (RNAi) pathways that silence gene expression

miRNAs

• 20-25 nucleotides in length

• similar to siRNAs because they participate in RNAi

• differs because they are synthesized from precursor molecules with complicated secondary structure

5 types of sncRNAs with more specialist roles

1. vault RNAs present in vault complex

2. 7SL RNA signal recognition particle (SRP)

3. 7SK RNA component of pTEF-b (positive elongation factor B)

4. Rnase P (involved in rRNA processing)

5. Telomerase (adds seq to end of chromosomes)

explain northern blotting

1. RNA or DNA on agarose gel with ³²P labelled markers

2. electrophoresis moves sample from anode to cathode

3. solution passes through gen and membrane to paper towels

4. DNA moves to the membrane only

5. membrane put into a seal-a-meal bag and hybridized to probes

6. unbound probe washed off

7. membrane exposed to X-ray film to visualize

what is the overall goal of differential display

looks at the difference of gene expression between two samples to be used for further study in disease research

explain differntial display in three steps

1. oligoT primer at high molarity so it can bind to all mRNA (get a bunch of cDNA after first strand synthesis)

2. short P primers are added which are desgined to have a good chnage of hybridizing to many sequences because it is only 6 nucleotide long

3. PCR is then done with the p primer and t primer (if this works, then a limited number of PCR products should be amplified that when separated on a gel, will give a snapshot of expression)

explain in one sentence what a microarray analysis is

hybridization to RNA or cDNA preparations to DNA chips which usually represents coding capacity of the genome

explain in 1-2 points what RNA-seq is

• relative amount of the transcripts in different transcriptomes are assed from the number of reads that map to the gene of interest

• genes whose expression are expected to be the same in the two tissues are used to normalize the data

explain 7 in steps how a 2 channel microarray can detect changes in gene expression

1. extract mRNA

2. make cDNA reverse trasncript and label them with fluorescent dyes

3. hybridize to the microarray

4. laser excitation at dye-specific wavelength

5. detect laser emission

6. computer calculates relative levels of hybridized probe

what is a DNA chip/DNA microarray

• collection of microscopic DNA spots attached to a solid surface

explain the probes or reporters in the microarray

• each DNA spot contains 10-12 moles of a specific DNA sequence known as probes or reporters

• can be short section of a gene or other DNA element that are used to hybridize to a cDNA or cRNA target

tetrahymena micronucleus characteristics

• diploid, divides by mitosis/ meiosis

• transcriptionally silent during growth

• 5 unique chromosomes

tetrahymena macronucleus characteristics

• divides by amitosis

• not inherits sexually

• 250 unique chromsomes

• 45 cops of each chromosome

• transcriptionally active

explain in steps tetrahymena instructions

1. pairing

2. meiosis 1 and 2

3. pre zygotic mitosis

4. pronuclear exchange

5. post zygotic mitosis x2

6. parental MAC elimination, daughter MAC development

7. exconjugant seperation, one daughter MIC eliminated, daughter MAC development

8. MIC mitosis, first fission MACs distributed to different kayonides

how did tetrahymena microarray become

• The first-generation predictions of ORFs based on the nearly complete sequence of the Tetrahymena thermophila macronuclear genome became available enabling microarray- based genome-wide analysis of mRNA abundance in different stages of the life cycle in this model organism

hierarchal clustering

comparing expression levels of the set of genes in transcriptome that have been analyzed and assigning a value that indicates the degrees of relatedness between those expression levels

heat map

uses colours to display the relative values of all tile within a given experimental condition

what is RNA seq

• sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing cellular transcriptome

what can RNA seq show

• alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments

what are the 2 methods used to assign raw sequence reads to genomic features

1. De novo assembly: does not require a reference genome to reconstruct the transcriptome, and is typically used if the genome is unknown, incomplete, or substantially altered compared to the reference

2. Mapped to Reference Genome: relies on the same methods used for DNA alignment, with the additional complexity of aligning reads that cover non-continuous portions of the reference genome

explain the steps for RNA seq

1. purify mRNA via oligo dT

2. make into RNA fragments or cDNA

3. create EST library with adaptors

4. create short sequence reads

5. get exonic reads from RF and coding sequence