ncRNA Structural 1.rRNA 2.tRNA 3.snRNA 4.snoRNA 5.cleavage: Rnases P & MRP, U3, snR30, etc Regulatory 1.Small siRNA miRNA 2.Long Activator Enhancer silencing
Other ncRNA? Incredible diversity of functions! Epigenetic Directly regulating transcription Post-transcriptional regulation Some are made by Pol II, others by Pol III
Our ncRNA 1.Extracted total RNA, separated out and sequenced nt
Our ncRNA 1.Extracted total RNA, separated out and sequenced nt 2.BLAST to find relatives (and discard tRNA, etc)
Our ncRNA 1.Extracted total RNA, separated out and sequenced nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find mutants
Our ncRNA 1.Extracted total RNA, separated out and sequenced nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find genes that weren’t near protein- coding genes
Our ncRNA 1.Extracted total RNA, separated out and sequenced nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find genes that weren’t near protein- coding genes 4.33 Hits: order seeds
Studying your ncRNA 1.Does it resemble anything known? Remember that many ncRNA bind known targets
Studying your ncRNA 1.Does it resemble anything known? Remember that many ncRNA bind known targets 2.Can it adopt a stable structure?
Studying your ncRNA 1.Does it resemble anything known? Remember that many ncRNA bind known targets 2.Can it adopt a stable structure? 3.Has its expression been detected by other means? MPSS, microarrays, etc
Studying your ncRNA 1.Design primers to identify homozygous plants Seeds are supplied as heteroygotes to avoid obligate lethals
Studying your ncRNA 1.Design primers to identify homozygous plants Seeds are supplied as heteroygotes to avoid obligate lethals 2.Design primers to detect your ncRNA 3.Grow seeds of your line, look for plants that look weird and collect their seeds.
Primer/probe design Stem-loop primers for short RNAs where only have enough info for one primer