END POINT PCR /SEMI QUANTITAVE PCR/REAL TIME PCR

Question 1

Endpoint PCR

Answer 1

• After all cycles of PCR
• Analysis after all cycles of PCR are completed
• One of the steps to perform gene expression analyses
• Results are analysed at plateau
• Not quantitative
• Results depend on number of cycles and concentration of template
• Analysis of data limited by gel sensivity depletion

Question 2

Semi quantitative PCR

Answer 2

Results are analysed before the plateau e.g 25 cycles rathern 30 cycles)

Not linear

Limited dynamic range

Analysis based on if comparison among samples , housekeeping gene is equally expressed in all cells

Question 3

Real time PCR (qpcr)

Answer 3

Measure and analyse product at every cycle
Quantitative -figures
Require a special instruments - RT instruments
Theres no additional sample handling
Broad dynamic range
Typically used <250bp to ensure efficient amplification
Product include Taq polymerase , dnTPS , Mgc12 , buffer , primers , Template

Question 4

How gene expression assesed using real time PCR ?

Answer 4

Conversion cells mRNA into CDNA using reaction substrate .

Studies involve comparing the amount of one gene (target) amount of transcript in a cell to another gene ( reffered /calibrater) amount of transcript

Question 5

Types of methods

Answer 5

Anova- used to compare differences in amplications effiices between genes . Asses variability between a set of samples withn an individual group vs entire group of samples .