Lab Practical- Real time qPCR

how many DNA duplexes are acquired after 4 cycles of PCR?

• every cycle- DNA amount doubles

• 2^n

• therefore 2^4= 16

how does NFkB become activated and how does it work?

• liberates p65-p52 dimers which allows it to travel to the nucleus

• allows it to target genes with NFkB binding sites

• makes pro inflammatory cytokines such as Il1b

what does BAY-11 do?

• it targets IKK beta activation

• it stops the subsequent degradation of IkB- so dimers cannot be liberated and blocks NFkB dependent signalling

steps of per for RNA analysis

• DNA template unwinds- denaturing

• the free 3’OH, targeted primer adds to this- annealing

• DNA polymerases and free nucleotides are added to extend the gene of interest- extension

• DNA copies are made

what is the Ct value? how does it work?

Ct value is a threshold number- usually when the exponential stage happens

higher Ct- lower amount of starting material

lower Ct- higher amount of starting material

how does SYBR green work?

• a dye that stains dsDNA

• signal increases exponentially with every cycle during PCR

how does TaqMan qPCR?

• Taqman probe is added- has a fluorochrome and quencher separated by short DNA sequence specific to the gene of interest

• probe binds gene of interest before replication

• as polymerase extends the primer, it encounters the probe

• polymerase TaqMan polymerase has exonuclease activity and degrades the probe- this releases the fluorphore from the quencher

• when fluorophore is no longer quenches- emits light

• each cycle- more probes are cleaved and fluorescence increases

how is dCT calculated?

• take Il1b value of 30 etc and subtract from housekeeping gene 16= 14

• take Il1b value with LPS treatment 20 from housekeeping gene 15= 20

how is ddCT value calculated?

• take dCT value from untreated - untreated: 14-14=0

• take dCT value from treated - untreated= 5-14=-9

how is relative quantification calculated?

• 2^-ddCT

• untreated= 2^-(0)

• treated= 2^-(-9)=512

• 512 fold increase in Il1b mRNA in LPS treated samples

how much of x2 RT master mix is needed to be added to a 20microL solution?

• 10 microL

if you get a 150 absorbance value for RNA from the spec, how many microL of it do you need to add?

convert 1 microL to 1000ng/ml

• if you get 150ng/ml=1.50microg/mL

• 1000/150=6.667

• 6.7microL of RNA

• 9microL-6.7= 2.3microL of water

therefore:

• 6.7microL of RNA

• 2.3microL of water

• 10microL of RT buffer

• 1microL of RT enzyme

• final vol of 20microL