RT-qPCR (Group 9) Flashcards
used to detect and quantify RNA
quantitative reverse transcription polymerase chain reaction (RT-qPCR)
used as template for quantitative PCR or real-time PCR reaction
total RNA or mRNA transcribed to complementary DNA (cDNA)
what is used to measure the amount of amplication product in each PCR cycle
fluorescence
where is RT-qPCR used
- gene expression analysis
- RNA validation
- microarray validation
- pathogen detection
- genetic testing
- disease research
Two types of RT-qpCR
- One-step RT-qPCR
- Two-step RT-qPCR
- both reverse transcription (RT) and quantitative PCR (qPCR) amplication are performed in a single tube in a single reaction mixture
- only utilizies sequence-specific primers
one-step RT-qPCR
reverse transcription (RT) and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies
two-step RT-qPCR
- preferred due to advantages such as requiring fewer purification steps, leading to more quantitative template recovery and faciliating better result normalization based on cell counts
- avoids potential biases arising from mRNA enrichment steps, ensuring consistent results across different mRNA targets
- most suitable for many applications since relatve quantification of targets is often prioritized over absolute sensitivity of detection
total RNA
offers slightly higher sensitivity compared to total RNA but may introduce complexities due to need for enrichment steps
mRNA
- designed to anneal to specific regions of the RNA transcript of interest
- useful when studying particular RNA species or when specific regions of RNA molecule needs to be targeted
- can provide higher specificity compared to random primers or oligo(dT) primers
Sequence-Specific Primers
- short oligonuleotide with a random sequence of nucleotides
- anneal nonspecifically to regions of the RNA template, allowing for the initiation of reverse transcription of multiple sites
- useful for synthesizing cDNA from a diverse population of RNA molecules, including mRNA, non-polyadenlyated RNA, and RNA with secondary structure
Random Primers
- allow for the capture of both polyadenylated mRNA transcripts (targeted by oligo(dT) primers) and non-polyadenylated RNA molecules (targeted by random primers)
- used in reverse transcription reaction
combination of Oligo(dT) and Random Primers
responsibel for converting RNA into DNA
reverse transcriptase (RT)
may be added to imporve qPCR efficiency
RNase H
for RT-qPCR, it is advantageous to choose what kind of reverse transcriptase?
with high thermal stability
plays a crucial role in the specificity and accurac of RT-qPCR
primer design
involes spanning exon-exon junctions to mitigate the risk of false positives from genomic DNA contamination
optimal primer design
imperative for discerning genuine RNA amplification from potential DNA contamination
minus reverse transcriptase control
Analytical steps of RT-qPCR
- isolate RNA
- anneal oligo(dT) primers
- first strand synthesis
- denaturation
- primer annealing and extension
- DNA synthesis and fluorescence detection
- initial step, aiming to extract RNA containing the target sequence
- must ensure free from contaminants that could hinder reaction
isolate RNA
anneal specficially to the poly(A) tail found at the 3’ end of most eukaryotic mRNA molecules
anneal oligo(dT) primers
- reverse transcription is initiated by the addition of reverse transcriptase enzyme, which synthesized a complementary DNA strand (cDNA) from the RNA template using the oligo(dT) primers
- converts the RNA template into single-stranded cDNA molecules
first strand synthesis
reaction mixture is heated to a high temperature to __ the RNA-cDNA hybrid molecules, separating the two strands and allowing the cDNA to become single-stranded
denaturation
- PCR primers specific to the target sequence are introduced into the reaction mixture
- primers bind to complementary sequences on the single-stranded cDNA template
- reaction is cooled to facilitate primer annealing to their target sequences
- subsequently, temperature is elevated to enable the DNA polymerase enzyme to extend the primers
- extension step leads to the synthsis of new DNA strands from the cDNA template
primer annealing and extension
- in each cycle of PCR, DNA polymerase synthesized new DNA strands from the cDNA template and primers
- fluorescently-labeled nucleotides are incorporated into the newly synthesized DNA strands during DNA synthesis
- fluorescence intensity increases in proportion to the amount of DNA synthesized
- real-time measurement of fluorescence by the PCR instrument after each cycle allows for target DNA quantificatin
- rise in fluorescence over successive cylces signifies exponential amplification of target sequence
DNA synthesis and fluorescence detection
application of RT-qPCR
detection of SARS-CoV-2 in the early stages of COVID-19
