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Recombinant DNA TechnologyCHMI 4226 E

Week of Jan 23, 2009

Qualitative and quantitative methods for the analysis of gene

expression

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cDNA clones!

Ori C

AmpR

Sequencing

Restriction mapping

Sub-cloning

IdentificationExpressed sequence tag

(EST)

mRNA expressionMutagenesis

Complete?

Blast

probe

5’/3’ RACE

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Gene expression

• 5’ untranslated region (5’UTR): – located upstream of the ORF– Generally short (<150b)– Involved in the regulation of the initiation of

translation

• Open reading frame (ORF):– The region of the mRNA encoding a

specific protein– Sequence is well conserved across

species

• 3’UTR:– Located downstream of the ORF– Generally quite long (>1000 b)– Involved in regulating mRNA stability as

well as translation

mRNA5’UTR ORF 3’UTR

AUG

UAAUGAUAG

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Gene expression• What would we like to know about our favorite mRNA?

– Size of the mRNA (that actually matters…..)

– Presence of alternative forms of the mRNA• Inclusion/exclusion of specific exons or introns (may affect the integrity or

coding potential of the ORF)• Longer/shorter 5’UTR or 3’UTR (would affect translation efficiency or mRNA

stability)

– Expression levels• During development• Tissue-specificity• Upon specific conditions (e.g. stress)• Pathological situations

– Which cells in a specific organ expresses our favorite mRNA

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Most widely used methods

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Northern Blot

• Qualitative

• Requires a nucleic acid probe

• Useful for:– Comparing the expression levels of a gene under different

conditions

– Determining the size of a particular mRNA

– Identifying alternative forms of an mRNA (alternative splicing)

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Northern blot analysis

RNA

RNARNA

RNA

RNA

RNA

RNA

RNA

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Northern BlotAnalysis of tissue-specific

expression

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Northern Blot

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Northern Blot – internal controls

• Detecting different levels of mRNA expression may be due to unequal RNA loading on the gel;

• To control for this possibility, the blot is also separately probed for a control mRNA, the expression of which does not change markedly under the conditions tested.

• Examples of control mRNAs: actin, GAPDH, 2-microglobulin

• Alternatively, a picture of the ethidium bromide-stained gel can be taken to visualize the rRNAs.

• The gel/autoradiogram can then be scanned, and the data represented as the signal intensity of the Favorite mRNA over the control.

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Northern BlotDetection of mRNA Induction

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Reverse transcription/PCR (RT-PCR)

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RT-PCR

• Steps:– 1. RNA isolation– 2. Conversion of mRNA into cDNA with reverse transcriptase– 3. Amplification of the desired target by PCR

• Advantages:– Extremely sensitive, very simple and fast method to analyse gene

expression;– Very little material is required (<ng vs >ug for Northern);– Multiplexing is possible: amplification of several different targets in a

single experiment;– Can yield quantitative data (i.e. # of mRNA molecules per cell).

• Drawbacks:– Too sensitive: false positives due to contamination is a real problem;– Obtaining quantitative data is difficult and requires a lot of optimization.

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PCR-Plateau effect

http://www.biotechlab.northwestern.edu/pe/sld021.htm

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PCR-Plateau effect

http://www.biotechlab.northwestern.edu/pe/sld021.htm

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PCR-Plateau effect• Plateau Effect - The plateau effect is an attenuation of the normally

exponential rate of product accumulation in a PCR reaction. It can be caused by:– depletion of dNTPs

– depletion of primers

– stability of the reactants (e.g. enzyme activity; particularly at the denaturation temperature)

– end product inhibition by duplex DNA

– non-specific competition for resources (production of incorrect product)

– reannealing of specific products to one another instead of to the primers (this is particularly problematic when product concentration is high).

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PCR-Plateau effect

http://www.biotechlab.northwestern.edu/pe/sld021.htm

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Semi-quantitative PCR

• In semi-quantitative PCR, the expression level of the favorite mRNA is expressed relative to an internal (part of the mRNA sample) or external (added for the PCR reaction) control.

• Problem: signal must be obtained during the linear phase of amplification.

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Semi-quantitative PCR

GAPDH

GADD45

GADD153

Culture Time 1 day 4 days 6 days 6 days -Gln +Gln

21 24 27 30 21 24 27 30 21 24 27 30 21 24 27 30

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Quantitative RT-PCR Competitive RT-PCR

• Problems with the use of controls for quantitative PCR:

– Differences in amplification efficiency due to:• Difference in size• Difference in nucleotide composition• Difference in primers used for amplification

(different Tms, ect)– Don’t know the absolute amount of internal

control in the reaction

• Mimick:– External control of known quantity– Size very similar to target (± 50 bp)– Same sequence as target– Uses same primers to amplify target and

mimick– Mimick will compete with target during the

amplification– Since we know the amount of mimick added

to the reaction, we can get the absolute amount of the mRNA of interest in our sample.

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Competitive RT-PCR

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Real-Time PCR

• Makes use of fluorescent probes allowing us to monitor the progress of the PCR reaction IN REAL TIME (i.e. no need for agarose gel electrophoresis)...

• Major advantages:– Allows one to rapidly terminate the PCR experiment if something

goes wrong;– Quantitative– Small volumes (microliter reactions): save a lot of $$$

• Major disadvantages:– Instrumentation– Getting quantitative data is not trivial

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Real-Time PCRTechnologies

TaqMan technology

SYBR Green technology

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Real-Time PCRInstrumentation

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Real-Time PCR

• The increase in fluorescence intensity is used to follow the progress of reaction.

• 10-fold dilutions of a DNA sample are amplified;

• The more DNA in the sample, the less cycles it takes to amplify it.

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Real-Time PCR• Specificity is cheched

by performing « melting curve » experiment with the final PCR product

All PCR products are identical

PCR product in « red » sample Is different than the others

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Real-Time PCR• After the experiment, the data is examined for the PCR

cycle at which a fluorescence signal above background is detected: this is called the cycle threshold, or Ct;

Threshold

Threshold

Log scale

Ct = 22

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Real-Time PCR• Quantification can be achieved by

amplifying known amounts of a DNA sample, and using the data to graph a standard curve of the Ct vs Log starting DNA concentration/ copy number;

• The amount of starting DNA in the unknown is then obtained using this standard curve;

Ct

Threshold

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RNAse Protection Assay (RPA)

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RNAse Protection Assay (RPA)

• Involves the liquid hybridization of an RNA probe to an RNA sample (more efficient/quantitative than solid phase hybridization [Northern]);

• Unhybridized RNAs are degraded with a mixture of RNAses; the hybridized RNA probe is protected from RNAse action

• Protected RNA probe is then fractionnated by gel electrophoresis and visualized by autoradiography/phosphorimaging

• Quatification is possible by cutting the bands from the gel and counting the amount of radioactivity using a scintillation counter.

Phosphorimager(pixels)

CPMs(scintillation counter)

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RNAse Protection Assay (RPA)

• The RNA probe is prepared by in-vitro transcription of a cDNA molecule in the presence of [32P]UTP;

• The probe can be designed to contain regions of sequence divergence between different forms of an mRNA: simultaneous detection of several products of alternative splicing;

• Multiplexing is possible: several probes detecting different mRNAs can be mixed an used in a single tube (i.e. internal controls).

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RNAse Protection Assay (RPA)

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RNAse Protection Assay (RPA)

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Hybridation In-situ

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In-situ Hybridization

• Allows the detection of a specific mRNA in whole cells/tissues/organs/animals;

• The probe is labeled with either a radioisotope or a fluorescent dye, and hybridized to the material of interest;

• Novel imaging techniques allow scientists to obtain spectacular data!

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In-situ Hybridization

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In-situ Hybridization

PNAS. 2003 100 (suppl. 1): 11836–11841

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In-situ Hybridization

SCIENCE. 2004. 305: p846