Measuring RNA transcript levels

Research problem – A small RNA, RteR, inhibits transfer of the 65 kb conjugative transposon CTnDOT attR attL ermF tetQ excision regulatory transfer oriT intDOT mob PcPc PEPE RteR PTPT 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

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 P tra A B C D E F G H I J K L M N O P Q traAtraB-CtraDtraE-FtraF-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

A typical plot from a qPCR run

Melt curves to ensure primer quality

Calculating when a sample crosses a given threshold (Ct or Cq) to quantify transcript abundance

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 ΩQAB pJW305 - Tc Empty Vector T7 promoter T7 promoter PCR In vitro transcription reaction + 32 P-αUTP Antisense ssRNA probe to region of interest

Decade Marker BT4001 ΩQAB pJW305 - Tc+ Tc Can detect multiple transcripts

RACE analysis to identify 5’ and 3’ ends of RteR Total RNA 5’ RACE 5’-P + TAP - TAP 5’-PPP 5’-P cDNA synthesis PCR + TAP- TAP 5’-PPP adapter ligation 3’ RACE OH-3’ calf. Int. phos. adapter ligation cDNA synthesis PCR processed end + TAP- TAP primary end