Gene expression Northern and RTPCR carla'gardnervanderbilt'edu - PowerPoint PPT Presentation
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Gene expression Northern and RTPCR carla'gardnervanderbilt'edu
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Assess the size, number, and relative abundance of gene transcripts ... anneal (hybridize) primers needed for DNA synthesis: 55 degrees ... – PowerPoint PPT presentation
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Title: Gene expression Northern and RTPCR carla'gardnervanderbilt'edu
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Gene expressionNorthern and
RT-PCRcarla.gardner_at_vanderbilt.edu
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Overview
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Northern blot analysis
- Assess the size, number, and relative abundance
of gene transcripts - RNA is size fractionated on a 1 agarose gel and
is then transferred to a positively charged nylon
membrane by positive-pressure alkaline transfer
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Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)
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Polymerase Chain Reaction (PCR)
- DNA amplification
- Basic cycle
- denature (melt) DNA 94 degrees
- anneal (hybridize) primers needed for DNA
synthesis 55 degrees - elongate new second strands 72 degrees
- repeat 30 times
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RT-PCR
- Makes DNA copy of mRNA then PCR amplifies
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Uses of RT-PCR
- mRNA detection and quantitation
- cloning
- cDNA library construction
- probe synthesis
- signal amplifications in in situ hybridizations
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Real Time-Polymerase Chain Reaction (RT-PCR)
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RT-PCR
- Developed to quantitate the expression of genes
- Key technique for analyzing purified RNA
- Quantitation is performed during amplification of
the cDNA - Dependent on reverse transcription condition
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RNase Protection Assay
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Uses of ribonuclease (RNase) protection assay
- Detecting and quantifying specific RNAs
- Gene and mRNA structure analysis
- Map transcription start and termination sites
- Subtle differences between the probe and target
RNA, such as mutational alterations, sequence
deletions or insertions
From Roche Molecular Biochemicals
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RNase Protection
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Advantages of RNase protection assay
- Large amounts of RNA probe can be synthesized by
the SP6/T7 or T3 transcriptional system - As little as 0.1 pg of mRNA can be detected
- Single-stranded probes hybridize specifically
only to their complementary strand - RNA hybrids with RNase result in fewer artifacts
than digestions of RNA-DNA with S1 nuclease - No background smear
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In Situ Hybridization and Immunohistochemistry
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In situ hybridization (ISH)
- Permits appearance of specific nucleic acid
sequences (genes) in their cellular environment - Hybridization of a labeled probe to a
complementary target sequence - Combined with immunocytochemistry, in situ can
relay gene activity information at the DNA, mRNA,
and protein level
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Immunohistochemistry
- Technique for identifying cellular or tissue
constituents (antigens)
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Luciferase assay
- Light is produced by converting the chemical
energy of luciferin oxidation through an electron
transition, forming the product molecule
oxyluciferin - Developed for reporter quantitation in mammalian
cells
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In Vitro Transcription
- Synthesis of RNA using a purified linear DNA
template - Uses
- blot hybridizations
- nuclease protection assays
- expression studies
- structural analysis
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In Vitro Translation
- in vitro synthesis of proteins in cell-free
extracts - identification of gene products
- localization of mutations through synthesis of
truncated gene products - protein folding studies
- incorporation of modified or unnatural amino
acids for functional studies.
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