L12: Analysing Gene Expression And Its Regulation Flashcards
Northern blots
Method for detecting amt of particular transcript
Run RNA in gel -> nucleic acids separated according to size through agarose-gel electrophoresis -> separated nucleic acids blotted onto nitrocellulose paper by suction of buffer through gel & paper -> radiolabelled probe hybridised to separate RNA -> labelled probe hybridised to complementary RNA -> bands visualised by autoradiography
Nitrocellulose
Tough, strong, charged -> absorb, DNA will stick to it
Pore size is fine (RNA will pass through but alkali and salt cant)
Absorbant blotting paper in northern blots
Absorb alkali solution and draw liquid through
Liquid from base -> bridge/sponge -> evenly through gel -> pull RNA molecules up from movement of solution onto nitrocellulose membrane -> RNA left on nitrocellulose membrane, alkali solution continue to pass through until all RNA attaches to nitrocellulose membrane
Probe
Ss and labelled nucleotides (chemical or radioactive labels aka tags)
Will bind complementary sequences on membrane
Fragment of DNA encoding part or all of YFG
Target in Northern Blots
mRNA (transcripts) from cells or tissues which could include transcript of YFG
Preparation of target in Northern Blots
Cellulose matrix with covalently attached oligo (dT) chains. (Bind anything with poly(A) tail -> connect go solid matrix. )Add solution of total RNA in NaCl
- > eukaryotic mRNA with poly(A) tails hybridise to oligo (dT) chains on cellulose. rRNA m, tRNA pass right through column. -> wash with NaCl to remove residual rRNA, tRNA -> salt will dissociate proteins from cellulose and help mRNA and DNA interaction
- > elute mRNA from column with water -> collect and evaluate mRNA solution
Preparation of probe in Northern Blots
Random priming: purified DNA fragment -> denature and anneal with mixture of hexanucleotides -> random hexamers bind to denatured DNA fragment -> use DNA pol to incorporate labelled nucleotides -> pop of DNA molecules that contain labelled examples of all sequences on both strands
Polynucleotide kinase: purified DNA restriction fragment -> add p32 ATP -> phosphorylate -> DNA labeled at 5’ ends with polynucleotide kinase -> restriction nuclease cuts DNA helix into 2 different sized fragments-> separated by electrophoresis-> desired DNA fragment with ss labeled at one end
Can use PCR: dsDNA -> heat to separate strands -> hybridisation of primers -> +DNA polymerase (dATP incorporated during action of Taq pol) -> DNA synthesis from primers. Detection of label- antibody with fluorescent tag against chemical used to label probe
Prehybridisation in Northern Blots
Blocks non-specific sites on membrane
Need to make sure binding is specific to sequence desired before adding probe to nitrocellulose membrane
Buffer contains ‘blocking agent’ -> block and bind to membrane. Skim milk powder, bovine serum albumin, genomic DNA from non related species (e.g. herring sperm DNA for blots of plant RNA)
Northern blot stringency (temp)
Detection of transcripts with sequences identical to that of probe: temp set below melting temp (Tm) of perfect double helix in hybridisation solvent (high stringency)
Detection of transcripts with sequences related to that of probe: lower temp (low stringency)
Manipulated at hybridisation and/or wash steps
Northern blot stringency (salt conc)
Detection of transcripts with sequences identical to that of probe: use low salt conc (high stringency)
Detection of transcripts with sequences related to that of probe: increase salt conc (lower stringency)
Usually manipulated at wash steps
in situ Hybridisation: Spatial & temporal examination of gene expression and regulation
Tissue: chemically preserved & embedded in wax. Sectioned (cut into thin slices, attach to slide and remove wax -> RNA molecules can be presented to probes) different from Northern- probing tissue with probe to see where transcript is.
Probe generation (not radioactive. Radiation goes into all directions -> resolution not great), prehybridisation, hybridisation (use chemical probe -> insoluble pink, purple product), washes, detection
Microarrays (gene chips): monitoring expression of thousand of genes simultaneously
Probe: represents gene made from mRNA
Isolate RNA from control and experimental cells or tissues
Convert mRNA to cDNA with reverse transcriptase and nucleotides labelled with fluorescent dye (target)
Mix labelled cDNA pop, denature, hybridise to genes (probes) attached to chip
Measure fluoresence at each spot on microarray. Comparison of fluoresence intensities give relative quantification
If not the colour of the dyes -> 1:1 (no change in gene expression. Same amt of each)
Microarrays: Quantitative measurement of thousands of genes simultaneously
Cluster analysis: groups genes that show similar expression patterns under given set of conditions
Allows identification of co-expressed and/or co-regulated genes
Reverse transcription quantitative PCR (RT-qPCR): measuring transcript abundance in real time
mRNA (reverse transcription)-> cDNA -> PCR with gene-specific primers
PCR products detected by fluorescent tags/dyes and specialised thermocycler. As PCR runs, amt of product increases -> fluorescent signal increases -> signal quantified
RT qPCR: quantifying transcript abundance
Generate standard curve. Do qPCR with known amt of target transcript = standards -> cloned cDNA or PCR product
As PCR runs, amt of product increases -> fluorescent signal increases
At one cycle in PCR, fluorescent signal for each standard crosses an amt threshold above background -> linear part of PCR
Plot Ct values of standards against log amt of standards
Do qPCR with standards and unknown sample. Plot standard curve. Use standard curve to determine initial conc of transcript for YFG
