RT-qPCR Flashcards
1
Q
method used to detect and measure RNA levels
A
quantitative reverse transcription PCR
2
Q
where is quantitative reverse transcription PCR used
A
- gene expression analysis
- pathogen detection
- cancer research
- drug development
3
Q
Basic Principle of the Technique
A
- reverse transcription (RT)
- quantitative PCR (qPCR)
4
Q
- total RNA or mRNA are transcribed into complementary DNA using the enzyme reverse transcriptase
- process stabilizes the form of DNA to be amplified
- serves as template for subsequent PCR amplification
A
reverse transcription (RT)
5
Q
- fluorescence is utilized to measure the quantity of amplification product at each cycle of the PCR reaction
- allows for real-time monitoring of DNA amplification, with the intensity of fluoresnce corresponding to the amount of DNA present
- can determine the abundance of target DNA sequence in the sample
A
quantitative PCR (qPCR)
6
Q
General Components of RT-qPCR
A
- template RNA
- sequence-specific primers
- reverse transcriptase
- DNA polymerase
- buffer
- deoxyribonucleotide triphosphates (dNTPs)
- fluorescent detection system
- real-time PCR machine
7
Q
Analytical Steps of RT-qPCR
A
- sample isolation
- reverse transcription
- real-time PCR
- data analysis
8
Q
sample isolation
A
- RNA isolation
- RNA integrity check
9
Q
RNA free from contaminants
A
RNA isolation
10
Q
ensures that isolated RNA is intact and suitable for further analysis
A
RNA integrity check
11
Q
reverse transcription
A
- cDNA synthesis
- One step/two step RT-qPCR
12
Q
- converting RNA to cDNA using reverse transcriptase enzyme
- oligo(dt) primers often used to initiate
A
cDNA synthesis
13
Q
combines RT and PCR to simplify process
A
one step RT-qPCR
14
Q
separate RT and PCR offering flexibility in design and optimization
A
two step RT-qPCR
15
Q
real-time PCR
A
- amplification
- fluorescence detection
- CQ determination (CT, cycle threshold)
