RT-qPCR (GROUP 7)

Question 1

a method used to detect and measure rna levels.

Answer 1

Quantitative reverse transcription

Question 2

• the total rna or mrna are transcribed into complementary dna using the enzyme reverse transcriptase
•this process stabilizes the form of dna to be amplified this serve as the template for subsequent pcr amplification.

Answer 2

Reverse transcription (RT)

Question 3

• fluorescence is utilized to measure the quantity of amplification product at each cycle of the pcr reaction.
• This method allows for the real time monitoring of dna amplification, with the intensity of fluorescence corresponding to the amount of dna present.
• By quantifying the fluorescent signals generated during each cycle, researchers can accurately determine the abundance of the target dna sequence in the sample

Answer 3

Quantitative pcr (qPCR)

Question 4

In qPCR, what is utilized to measure the quantity of amplification product at each cycle of the pcr reaction.

Answer 4

fluorescence

Question 5

General components of RT-qPCR

Answer 5

1. Template rns
2. Sequence-specific primers
3. Reverse transcriptase
4. Dna pol
5. Buffer
6. Deoxyribonucleotide triphosphates
7. Fluorescence detection system
8. Real-time PCR machine

Question 6

Analytical steps:

Answer 6

1. Sample isolation
2. Reverse transcription
3. Real-time PCR
4. DATA ANALYSIS

Question 7

rna free from contaminants ( can be achieved through lysis, homogenization, purification)

Answer 7

Rna isolation

Question 8

insurance that isolated RNA is intact and suitable for further synthesis (can be achieved using gel electrophoresis and bioanalyzer)

Answer 8

RNA integrity check

Question 9

• Converting RNA to cDNA using reverse transcriptase enzyme.
• Oligo (dt) primers often used to initiate cDNA synthesis

Answer 9

cDNA synthesis

Question 10

often used to initiate cDNA synthesis

Answer 10

Oligo (dt) primers

Question 11

combines RT and PCR to simplify the process.

Answer 11

One step RT-PCR

Question 12

separate RT and PCR offering flexibility in design and optimization

Answer 12

Two step RT-PCR

Question 13

cDNA sequence is amplified undergoing cycles such as denaturation, annealing and extensions.

Answer 13

Amplification

Question 14

fluorescence signals are detected by intercalating dyes and hydrolysis probes.

Answer 14

Fluorescence Detection

Question 15

fluorescence exceeds the threshold, indicating exponential amplification

Answer 15

CQ Determination (CT, cycle threshold)

Question 16

lower CQ, higher starting target molecule copy numbers in the sample.

Answer 16

CQ Value Interpretation-

Question 17

comparing CQ values of the target gene to a reference gene allows for relative gene expression analysis

Answer 17

Relative Quantification

Question 18

determine number of target copies in the sample using standard curve of known target molecule concentration.

Answer 18

Absolute Quantification

Question 19

Steps in sample isolation

Answer 19

RNA isolation
RNA integrity check

Question 20

Steps in Reverse transcription

Answer 20

cDNA synthesis
One step/Two step RT-PCR

Question 21

Steps under Real-time PCR

Answer 21

Amplification
Fluorescence Detection
CQ Determination (CT, cycle threshold)

Question 22

DATA ANALYSIS

Answer 22

CQ Value Interpretation
Relative Quantification
Absolute Quantification

Question 23

Analytical steps
(RT-PCR)

Answer 23

1. Isolates rna
2. Anneal oligo (dt) primers
3. First strand synthesis
4. First cycle pcr synthesis
5. Pcr amplification

Question 24

Quantitative real tim- PCR (qPCR) ANALYTICAL STEPS

Answer 24

1. Isolates dna
2. Denaturation
3. Primer annealing and extension
4. Dna synthesis and fluorescence detection

Question 25

Reverse transcription quantitative real-time pcr (RT-qPCR) Analytical steps

Answer 25

1. Isolates rna
2. Anneal oligo (dt) primers
3. First strand synthesis
4. Denaturation
5. Primer annealing and extension
6. Dna synthesis and fluorescence detection