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Net-seq
- Nascent Transcript Sequencing
- Captures actively transcribed RNAPIIs on a column
- The RNAPII elongation complex is immuno precipitated and RNA extracted
- Extracted RNA is reverse transcribed to cDNA
- Sequencing of the cDNA allows us to see nascent RNA captured at the 3' end
- Can see what transcripts are being translated at the time of your experimental conditions
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Nuclear Run-on transcription
- measures how much RNA for a particular gene is made within a sample
- nucleic sample is purified and incubated (chilled) with a radioactive label that is incorporated into the NRA
- RNA is hybridized to a probe which corresponds to GOI
- radiation level determines quantity of RNA
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RNA-seq
- only detects levels RNA in a specific condition, which then you can compare
- needs fragments 200-800 nt long
- does not detect change in transcription
- analyzes which genes are on/off and to what extent
- first makes RNA to cDNA, add an adapter for targeted sequences
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Northern Blot Hybridization
- Isolate RNA and run it on a gel
- transfer it to a membrane and incubate it to your probe of interest and visualize it
- semi quantitative: compare fold change
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QPCR
- used to determine level of gene expression
- fluoresence is quantified but is not absolute
- measures fold change
- needs a standard curve to normalize teh data, created from known concentrations of your sample
- report CT values
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CT values
the number of cycles needed to replicate enough DNA/RNA to be detectable
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NanoStrings
- a direct counting method
- you have two probes, one gives a barcode for the gene of interest, other probe captures and tags with biotin
- sample is purified
- then immobilized with an electric current
- flourescent barcodes are imaged and counted
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NanoString Spatial molecular imaging
- place tissue sample on a slide that are targets for hybridization
- hybridize RNA specific probes and antibodies (biotin)
- assemble the flow cell
- visualize where the barcodes are localized within the tissue sample
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ddPCR
- droplet digital PCR
- PCR sample is emulsified in oil, which produces appx 20,000 nL droplets
- droplets have different quantity of RNA within
- fluorescence occurs if the target sequence and the cells are counted for fluorescence observed
- This is a Poisson distribution and can give absolute quantification of your sample
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in situ hybridization
- captures spatial/temporal expression information
- analyzes transcription level of a specific transcript
- Tissue is fixed and permeated to allow labeled probes to enter then visualized
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Reporter genes and fusion types
- Transcriptional fusions
- Translational fusions
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Transcriptional fusion
- see where the gene is transcribed into RNA
- tag uses the genes promoter
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Translational fusion
- You see where the gene is translated into proteins
- tag is following the gene sequence on the DNA
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Gal4 system
- Gal4 is a transcription factor that is a positive regulator of gene expression in galactose induced genes
- recognizes genes with a UAS to activate
- can be tissue specific
- used in many systems like Y2H, optogenetics
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Oligonucleotide microarrays
- oligonucleotides fixed to an array
- can fabricate them with direct spotting, inkject, and photolitography
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Which types of oligonucleotide arrays are created via photolithoraphy?
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cDNA-AFLP
- amplified fragment length polymorphism
- transcript profiling
- does not require sequence information
- can be used for gene discovery
- looks at differences in and patterns between control and experimental sets
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How do you isolate mRNA from all other types of RNA?
- extract RNA only exhibiting poly(A) tails
- oligo dT
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Akron seq
- captures 3' and 5' ends of mRNA concurrently
- keeps capped, polyadenylated mRNA
- ends are ligated with adapters and fragmented
- cDNA library created, amplified, sequenced
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NGS for direct sequencing/ RNA-seq
- Oxford Nanopore
- PacBio
- directly reads RNA and structural changes that might exist, like methylated Cs
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What is a software used to map reads to a reference genome?
Bowtie
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Which plot visualizes transcripts by plotting magnitude of change (fold change) vs statistical significance (p-values)?
Volcano plot
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Functional Genomics
- involves finding relationships between a collection of genes
- group the genes by type, sequence similarity, function similarity
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Genome Ontology
- This system exhibits a hierarchy with three levels
- biological process, cellular location, molecular function
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