Chapter_10_-_Analysis_of_Gene_Expression

Chapter_10_-_Analysis_of_Gene_Expression - 10. Amount of a...

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10.1 Analysing Transcription Amount of a specific mRNA at a point in time is dictated by the level of transcriptional activity and also the stability of the mRNA mRNA abundance does not necessarily correlate with the amount of protein
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10.1 Analysing Transcription 10.1.1 Northern blots In a Northern blot , total RNA or mRNA is separated on a electrophoretic gel under denaturing conditions (with formaldehyde) Visible bands for ribosomal RNA appear with total RNA in a gel lane Like the Southern blot and library screening, the mRNA is transfered onto a membrane filter; RNA bands are identified by hybridization with a labeled DNA probe onto the RNA-binding filter Figure 10.1 Northern blotting
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10.1 Analysing Transcription 10.1.2 RNase protection assay Solution instead of filter hybridization Labeled antisense RNA will bind to specific mRNA that you want to measure, forming dsRNA When RNase is added, all single stranded RNA degrades, except for the protected and labeled double-stranded hybrids More sensitive and quantitative than Northern blotting More tolerant of partial degradation Unlike Northern blotting, unable to determine the size of the transcript using this method Figure 10.2 Production of antisense RNA Figure 10.3 RNase protection assay
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10.1 Analysing Transcription 10.1.3 Reverse transcription PCR First strand cDNA synthesis of mRNA serves as a template in the PCR Extremely sensitive technique: possible to detect a single mRNA transcript from a single cell RT-PCR requires much less mRNA than a Northern blot not suitable for quantifying mRNA levels Figure 10.4 Reverse transcription PCR
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13.1 Analysing Transcription Reverse transcription PCR – Real time RT-PCR PCR carried out in the presence of a fluorescent dye ( Syber Green ) that intercalates with double stranded DNA A real-time machine carries out the temperature ramping of a typical PCR machine but will also monitor the fluorescence incorporated into each PCR reaction in real- time. Extrapolation of the fluorescence curve back to zero, gives a relative estimate of the starting amount of cDNA template also referred as the cycle threshold (or C T ) A lower C T value (such as 20) represents more template than C T = 22 or 25. Real-time results are standardized to parallel RT-PCR data gathered using, constituitively expressed, housekeeping genes which are expected not to be regulated Figure 13.5 Real-time PCR
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10.1 Analysing Transcription 10.1.4  In situ  hybridization Besides solution and filter hybridization, DNA/RNA probes can hybridize to
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This note was uploaded on 07/10/2010 for the course BIOL 208 taught by Professor Chuong during the Spring '09 term at Waterloo.

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Chapter_10_-_Analysis_of_Gene_Expression - 10. Amount of a...

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