RT-qPCR (Group 9)

Question 1

used to detect and quantify RNA

Answer 1

quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Question 2

used as template for quantitative PCR or real-time PCR reaction

Answer 2

total RNA or mRNA transcribed to complementary DNA (cDNA)

Question 3

what is used to measure the amount of amplication product in each PCR cycle

Answer 3

fluorescence

Question 4

where is RT-qPCR used

Answer 4

• gene expression analysis
• RNA validation
• microarray validation
• pathogen detection
• genetic testing
• disease research

Question 5

Two types of RT-qpCR

Answer 5

1. One-step RT-qPCR
2. Two-step RT-qPCR

Question 6

• 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

Answer 6

one-step RT-qPCR

Question 7

reverse transcription (RT) and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies

Answer 7

two-step RT-qPCR

Question 8

• 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

Answer 8

total RNA

Question 9

offers slightly higher sensitivity compared to total RNA but may introduce complexities due to need for enrichment steps

Answer 9

mRNA

Question 10

• 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

Answer 10

Sequence-Specific Primers

Question 11

• 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

Answer 11

Random Primers

Question 12

• 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

Answer 12

combination of Oligo(dT) and Random Primers

Question 13

responsibel for converting RNA into DNA

Answer 13

reverse transcriptase (RT)

Question 14

may be added to imporve qPCR efficiency

Answer 14

RNase H

Question 15

for RT-qPCR, it is advantageous to choose what kind of reverse transcriptase?

Answer 15

with high thermal stability

Question 16

plays a crucial role in the specificity and accurac of RT-qPCR

Answer 16

primer design

Question 17

involes spanning exon-exon junctions to mitigate the risk of false positives from genomic DNA contamination

Answer 17

optimal primer design

Question 18

imperative for discerning genuine RNA amplification from potential DNA contamination

Answer 18

minus reverse transcriptase control

Question 19

Analytical steps of RT-qPCR

Answer 19

1. isolate RNA
2. anneal oligo(dT) primers
3. first strand synthesis
4. denaturation
5. primer annealing and extension
6. DNA synthesis and fluorescence detection

Question 20

• initial step, aiming to extract RNA containing the target sequence
• must ensure free from contaminants that could hinder reaction

Answer 20

isolate RNA

Question 21

anneal specficially to the poly(A) tail found at the 3’ end of most eukaryotic mRNA molecules

Answer 21

anneal oligo(dT) primers

Question 22

• 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

Answer 22

first strand synthesis

Question 23

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

Answer 23

denaturation

Question 24

• 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

Answer 24

primer annealing and extension

Question 25

• 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

Answer 25

DNA synthesis and fluorescence detection

Question 26

application of RT-qPCR

Answer 26

detection of SARS-CoV-2 in the early stages of COVID-19