qPCR detailed explanation Flashcards
qPCR is able to
amplify and quantify DNA concentration in cells
what is used to quantify DNA
fluorescent signals given off by DNA binding dyes
application of qPCR
- quantitative analysis of gene expression
- quantifying viral, bacterial and fungal loads e.g. if someones infected and how infected they are
what dye is used
STBR green base dye
SYBR
its fluorescent signal when it binds to double stranded DNA
when is fluorescence measured and why
at the end of the elongation step- since DNA is double stranded at this point
SYBR can only bind to
double stranded DNA
basic reasoning behind qPCR
the more DNA in the original sample, the more the sample will fluoresce
PCR can only work on
DNA
what must be used on mRNA
reverse transcriptase to produce cDNA- so PCR process will work
example of qPCR
if looking at a normal cell vs a cancerous cell- primers for RAS cDNA (converted from mRNA by RT) will be added to make sure the PCR is specific to the RAS mRNA. then the quantity of of mRNA in a cancerous cell can be compared to a normal cell. e.g. the cancerous cell sample should fluoresce more- needing less cycles to fluoresce more due to a higher starting concentration
on the graph what is the dependent variable
cycle number
on the graph what is the independent variables
fluorescence
why must we use a control
to see a relative increase in certain genes (e.g. RAS) over other genes
what gene is often used as a control
GADPH- because expression shouldn’t change between cancer and non-cancer cells
how is the control used
a separate qPCR reaction is carried out and used as a comparison to the gene of interest
what does Ct stand for
cycle threshold
what is the Ct
the number of cycles required for the fluorescent signal to cross the threshold (exceed background levels)
Ct and its relationship with DNA
Ct is inversely proportional to the amount of target nuclei acid e.g. the lower Ct the more DNA/ mRNA in the original sample
calculation of difference expression using the graph
1) compare the control (GADPH) in normal vs cancer cells (record the change in Ct (cycle threshold) e.g. Ct= 10 (cancer); ct= 11 (normal). change in Ct= 11-10=1
2) then compare the RAS signal in normal and cancer cells. e.g. ct=21 (cancer); ct=26 (normal). change in ct= 26-21= 5
3) 5-1= 4, therefore 2^4= 16
4) 16 fold increase in RAS expression in cancer cells
can be used in
diagnoistics- e.g. how much virus we have
-if we don’t know what genes we are looking for then we need to use RNA sequencing
method for how we would qPCR to check if RAS is being over expressed e.g. based on [mRNA]
- PCR only works on DNA
- mRNA - RT–> cDNA
- using primers for RAS carry out PCR (denature, annexing, extension)
- add dye which fluoresces when it attaches to dsDNA
