Expression of the Expression of the GGeenomenome

The transcriptomeThe transcriptome

Decoding the Genetic Decoding the Genetic InformationInformation

Information encoded in nucleotide Information encoded in nucleotide sequences contained in discrete sequences contained in discrete unitsunits The genesThe genes

The information contained in the The information contained in the genes is transcribed to generate genes is transcribed to generate the RNAs and then decoded to the RNAs and then decoded to generate the proteinsgenerate the proteins

The GenesThe Genes

Transcription initiation site

Termination sequence

3’

Promoter/Regulatory sequences

5’

Introns

RNA Transcript

5’ untranslated region3’untranslated region

Exon 2 Exon 3Int. 2Exon 1 Int. 1

Exons

Only one of the two strands is coding!

CodingCoding

Coding strandCoding strand Positive strandPositive strand Sense strandSense strand Strand which is complementary to the Strand which is complementary to the

template strandtemplate strand Strand of which the sequence is the Strand of which the sequence is the samesame as that of the RNA transcript as that of the RNA transcript

Strand on which is located the Strand on which is located the promoterpromoter

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Non CodingNon Coding

Non coding strandNon coding strand Negative strandNegative strand Antisense strandAntisense strand Template strandTemplate strand Strand of which the sequence is Strand of which the sequence is complementarycomplementary to that of the to that of the RNA transcriptRNA transcript

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Codant Vs Non-codingCodant Vs Non-coding

DNA: 5’ TAG 3’3’ ATC 5’

Translation

LeuProtein:

Genetic code : CUA = Leu UAG = Stop

Transcription

RNA: ?5’ 3’

GenomeGenome

TranscriptomeTranscriptomeCollection of RNA from genes that code

for proteinsCollection of RNA that represents the

fraction of the genome that is expressed

ProteomeProteomeCollection of proteins derived from

the transcriptome

One Genome

Is the transcriptome the same in all the cells of an organism?

Is the transcriptome always the same in a given cell?

Does a Sequence Code for Does a Sequence Code for a Transcript?a Transcript?

Northern Hybridization Analysis Northern Hybridization Analysis  RT-PCR RT-PCR 

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Comparaison of MethodsComparaison of Methods

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Northern RT-PCR

Sequence must be known

Presence or absence of a transcript

Allows to determine size

Sensitivity

Compare relative abundance

Obtain sequence of transcript

Determine which strand is transcribed

Determine how many transcripts are made from a single sequence

No Yes

Yes Yes

Yes No

Low High

Yes Yes

No Yes

Yes Yes

Yes No

THE SEQUENCE MUST BE EXPRESSED YES YES

Northern Analysis Northern Analysis

Isolate total RNA from cells or Isolate total RNA from cells or tissuetissue

Separate RNA according to their Separate RNA according to their sizes on denaturing agarose gelsizes on denaturing agarose gel Formaldehyde + FormamideFormaldehyde + Formamide

Hybridization with complementary Hybridization with complementary probeprobe rRNA

tRNA

Northern Hybridization Northern Hybridization

Requires a probeRequires a probe Hybridization= the probe has Hybridization= the probe has

sequences of the genesequences of the gene The sequence is expressedThe sequence is expressed

Intensity of hybridization signal Intensity of hybridization signal = relative abundance = relative abundance

Number of hybrids= number of Number of hybrids= number of transcriptstranscripts

Possibly number of genesPossibly number of genes12

Northern Hybridization Northern Hybridization

Allows to compare the relative Allows to compare the relative quantity of a transcriptquantity of a transcript Low sensitivityLow sensitivity Requires an internal controlRequires an internal control

Gene whose abundance is constant under the Gene whose abundance is constant under the different conditions examineddifferent conditions examined

– Controls for variations in the amount of RNA loadedControls for variations in the amount of RNA loaded– Use housekeeping genes :Use housekeeping genes :

Genes that ensure indispensable functions for Genes that ensure indispensable functions for the survival of all cell types the survival of all cell types

Constitutive expressionConstitutive expression13

NormalizationNormalization

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ProblemProblem

A Northern of ARN isolated from A Northern of ARN isolated from different tissues was probed with the different tissues was probed with the Fos gene as well as a house keeping Fos gene as well as a house keeping gene; Actin. Explain the results gene; Actin. Explain the results obtainedobtained

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Tissues: F C R P Tissues: F C R P

ActinActin

FosFos

RT-PCRRT-PCR

Allows the amplification of an RNA Allows the amplification of an RNA sequencesequence Isolate total RNA from cells or tissuesIsolate total RNA from cells or tissues Transcribe RNA into cDNA with Transcribe RNA into cDNA with

reverse transcriptasereverse transcriptase Amplify sequence of interest by PCRAmplify sequence of interest by PCR

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Reverse Transcriptase Reverse Transcriptase ReactionReactionGene Non-SpecificGene Non-Specific

17AAAAAAATTTT

AAAAAAATTTT

AAAAAAATTTTAAAAAAATTTTAAAAAAATTTT

Annealing of polyT primer

CollectionCollection of complementary DNAs to RNAs expressed at a given time under given conditions

AAAAAAA

AAAAAAAAAAAAAA

AAAAAAA

AAAAAAA

mRNA

AAAAAAATTTT

AAAAAAATTTT

AAAAAAATTTTAAAAAAATTTTAAAAAAATTTT

Transcription to cDNA

RT

Reverse Transcriptase Reverse Transcriptase ReactionReactionGene SpecificGene Specific

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AAAAAAA

AAAAAAA

AAAAAAA

AAAAAAA

AAAAAAA

Synthesis of cDNA

RT

DNA complementary to one one mRNA of interest

AAAAAAA

AAAAAAA

AAAAAAA

AAAAAAA

AAAAAAA

Annealing of gene specific primer

AAAAAAA

AAAAAAAAAAAAAA

AAAAAAA

AAAAAAA

RTRT PCR PCR

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cDNA Collection cDNA of mRNA of interest

Analysis on gel

PCR with primers specific to sequence of interest

RT-PCRRT-PCR

The sequence must be known in to The sequence must be known in to design primersdesign primers Amplification product =Amplification product =

The primer sequences are part of the geneThe primer sequences are part of the gene The sequence is expressedThe sequence is expressed

Intensity proportional = relative abundanceIntensity proportional = relative abundance The size of the amplification product is not The size of the amplification product is not

equal to the size of the transcriptequal to the size of the transcript

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