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Research problem
– A small RNA, RteR, inhibits transfer of the 65 kb conjugative transposon CTnDOT
attR attL
ermF tetQexcision regulatory transfer
oriTintDOT
mob
PcPE
RteR
PT
Question: precisely where in the transfer region is RteR binding???
Possible means of measuring transcript levels
• Promoter fusions
• Qualitative RT-PCR
• Quantitative or “real time”: RT-qPCR
• Northern blotting analysis
• 5’ and 3’ RACE – lengths of transcript rather than abundance
Promoter fusions
• A reporter gene (e.g. lacZ) is fused to a promoter of interest to test for transcriptional activity
• This represents only activity of promoter, and thus excludes other levels of transcriptional regulation
lacZ-35 -10
Reverse transcriptase (RT)-PCR
• A.k.a. qualitative or endpoint
• RT is “reverse transcriptase”: synthesize cDNA from total RNA pools– Generally isolate RNA from different cell
treatments, e.g.:• Carbohydrate source• Aerobic vs. anaerobic growth• Growth phase• With and/or without gene of interest in trans
Transcript levels +/- small RNA, RteR
Ptra
A B C D E F G H I J K L M N O P Q
traA traB-C traD traE-F traF-G
- + - + - + - + - +RteR
Quantitative or “real time” PCR
• Gives you information in real time after each cycle
• For obvious reasons a quantitative value, rather than a subjective relative value, can be more interesting and meaningful
Caveats to qPCR
• Experimental methods/calculations can greatly skew results, as with any technique
• Many have false sense of trust in the word “quantitative” believe technique is inherently more accurate
Northern blotting
• Another means of (literally) visualizing transcript levels
• Can detect RNA processing events that would other wise be unnoticed in RT-PCR
• Caveat is that it is qualitative, and there is a limit to transcript size due to electrophoresis capabilities
RteR Northern blotting analysis
Marker
BT4001 ΩQABpJW305- Tc
EmptyVector
150
100
90
8070
60
50
40
30
T7 promoter
T7 promoter
PCR
In vitro transcriptionreaction
+
32P-αUTP
Antisense ssRNA probe to region of interest
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