RT-qPCR Flashcards
method used to detect and measure RNA levels
quantitative reverse transcription PCR
where is quantitative reverse transcription PCR used
- gene expression analysis
- pathogen detection
- cancer research
- drug development
Basic Principle of the Technique
- reverse transcription (RT)
- quantitative PCR (qPCR)
- 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
reverse transcription (RT)
- 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
quantitative PCR (qPCR)
General Components of RT-qPCR
- template RNA
- sequence-specific primers
- reverse transcriptase
- DNA polymerase
- buffer
- deoxyribonucleotide triphosphates (dNTPs)
- fluorescent detection system
- real-time PCR machine
Analytical Steps of RT-qPCR
- sample isolation
- reverse transcription
- real-time PCR
- data analysis
sample isolation
- RNA isolation
- RNA integrity check
RNA free from contaminants
RNA isolation
ensures that isolated RNA is intact and suitable for further analysis
RNA integrity check
reverse transcription
- cDNA synthesis
- One step/two step RT-qPCR
- converting RNA to cDNA using reverse transcriptase enzyme
- oligo(dt) primers often used to initiate
cDNA synthesis
combines RT and PCR to simplify process
one step RT-qPCR
separate RT and PCR offering flexibility in design and optimization
two step RT-qPCR
real-time PCR
- amplification
- fluorescence detection
- CQ determination (CT, cycle threshold)
cDNA seqeunce is amplified undergoing cycles such as denaturaiton, annealing, and extensions
amplification
fluorescence signals are detected by intercalating dyes and hydrolysis probes
fluorescence detection
fluorescence exceeds the threshold, indicating exponential amplification
CQ determination (CT, cycle threshold)
data analysis
- CQ value interpretation
- relative quantification
- absolute quantification
lower CQ, higher starting target molecule copy numbers in the sample
CQ value interpretation
comparing CQ values of the target gene to a reference gene allows for relative gene expression analysis
relative quantification
determine number of target copies in the sample using standard curve of konwn target molecule concentration
absolute quantification
RT-qPCR application exapmle
study drug-induced changes of gene expression in tumor cell lines
