1. Measuring RNA transcript levels

2. 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???

3. 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

4. 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

5. 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

7. 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

8. 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

9. A typical plot from a qPCR run

10. Melt curves to ensure primer quality

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

12. 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

13. 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

14. RteR Northern blotting analysis Marker BT4001 ΩQAB pJW305 - Tc Empty Vector 150 100 90 80 70 60 50 40 30 T7 promoter T7 promoter PCR In vitro transcription reaction + 32 P-αUTP Antisense ssRNA probe to region of interest

15. Decade Marker BT4001 ΩQAB pJW305 - Tc+ Tc 150 100 80 60 40 Can detect multiple transcripts

16. 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