L8-9 Reverse Transcription

Question 1

Reverse transcriptase PCR (RT-PCR)

Answer 1

The most sensitive technique for mRNA detection and quantification from a single cell

Question 2

RT PCR first reaction

Answer 2

RNA, cDNA hybrid

1) denatured (annealing)
2) Extension - Taq polymerase
3) Primer (2-30 cycles)

Common controls
Eukaryotic : Actin
Prokaryotic : sigma70

Question 3

Quality control in RT-PCR

Answer 3

PCR will amplify cDNA and contaminate chromosomal DNA
RNA is treated with DNAse I prior to cDNA synthessis

Exon-Intron complex
Primer bind to exons
A cDNA is produced
PCR is used

Question 4

House keeping gene

Answer 4

Not affected by external sources, used to normal samples, it allows to see that the RNA samples are the same.
Used in RT-PCR

Question 5

Quantification of PCR (Radioactive labelling)

Answer 5

Radioactive nucleotides are used for PCR
Electrophorised
Look at the intensity of radioactivity (phosphorimager)

Ethidium bromide