qPCR detailed explanation

Question 1

qPCR is able to

Answer 1

amplify and quantify DNA concentration in cells

Question 2

what is used to quantify DNA

Answer 2

fluorescent signals given off by DNA binding dyes

Question 3

application of qPCR

Answer 3

• quantitative analysis of gene expression

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

Question 4

what dye is used

Answer 4

STBR green base dye

Question 5

SYBR

Answer 5

its fluorescent signal when it binds to double stranded DNA

Question 6

when is fluorescence measured and why

Answer 6

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

Question 7

SYBR can only bind to

Answer 7

double stranded DNA

Question 8

basic reasoning behind qPCR

Answer 8

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

Question 9

PCR can only work on

Answer 9

DNA

Question 10

what must be used on mRNA

Answer 10

reverse transcriptase to produce cDNA- so PCR process will work

Question 11

example of qPCR

Answer 11

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

Question 12

on the graph what is the dependent variable

Answer 12

cycle number

Question 13

on the graph what is the independent variables

Answer 13

fluorescence

Question 14

why must we use a control

Answer 14

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

Question 15

what gene is often used as a control

Answer 15

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

Question 16

how is the control used

Answer 16

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

Question 17

what does Ct stand for

Answer 17

cycle threshold

Question 18

what is the Ct

Answer 18

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

Question 19

Ct and its relationship with DNA

Answer 19

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

Question 20

calculation of difference expression using the graph

Answer 20

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

Question 21

can be used in

Answer 21

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

Question 22

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

Answer 22

• 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