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Flashcards in qPCR detailed explanation Deck (22)
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1
Q

qPCR is able to

A

amplify and quantify DNA concentration in cells

2
Q

what is used to quantify DNA

A

fluorescent signals given off by DNA binding dyes

3
Q

application of qPCR

A
  • quantitative analysis of gene expression

- quantifying viral, bacterial and fungal loads e.g. if someones infected and how infected they are

4
Q

what dye is used

A

STBR green base dye

5
Q

SYBR

A

its fluorescent signal when it binds to double stranded DNA

6
Q

when is fluorescence measured and why

A

at the end of the elongation step- since DNA is double stranded at this point

7
Q

SYBR can only bind to

A

double stranded DNA

8
Q

basic reasoning behind qPCR

A

the more DNA in the original sample, the more the sample will fluoresce

9
Q

PCR can only work on

A

DNA

10
Q

what must be used on mRNA

A

reverse transcriptase to produce cDNA- so PCR process will work

11
Q

example of qPCR

A

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

12
Q

on the graph what is the dependent variable

A

cycle number

13
Q

on the graph what is the independent variables

A

fluorescence

14
Q

why must we use a control

A

to see a relative increase in certain genes (e.g. RAS) over other genes

15
Q

what gene is often used as a control

A

GADPH- because expression shouldn’t change between cancer and non-cancer cells

16
Q

how is the control used

A

a separate qPCR reaction is carried out and used as a comparison to the gene of interest

17
Q

what does Ct stand for

A

cycle threshold

18
Q

what is the Ct

A

the number of cycles required for the fluorescent signal to cross the threshold (exceed background levels)

19
Q

Ct and its relationship with DNA

A

Ct is inversely proportional to the amount of target nuclei acid e.g. the lower Ct the more DNA/ mRNA in the original sample

20
Q

calculation of difference expression using the graph

A

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

21
Q

can be used in

A

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

22
Q

method for how we would qPCR to check if RAS is being over expressed e.g. based on [mRNA]

A
  • 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