Gene Cloning

The Analysis of the Regulation of Gene Expression 329
(a)
5'
mRNA – gene 1
3' 5'
5'
mRNA – gene 2
5'
3'
3' 5'
3'
mRNA – gene 3
3' 5'
3'
Random hexanucleotide
primer
Reverse transcriptase + dNTPs
5'
3'
5'
3'
3'
5'
cDNA – gene 2
3'
5'
cDNA – gene 1
5'
3'
5'
cDNA – gene 3
RNAse H
Store
3'
(b)
3'
5'
cDNA – gene 2
5' 3'
3'
3' 5'
cDNA – gene 1
3'
cDNA – gene 3
5'
5'
Quantitative real-time
PCR with primers
specific for gene 1
Gene specific
product is
amplified and
quantified
Figure 11.6 Quantitative real-time reverse transcriptase-PCR. a) RNA is
purified and random hexanucleotides are used to prime the synthesis of cDNAs
by reverse transcriptase for all RNAs present in the starting sample. RNA is
removed by RNAse H and the samples stored. b) The amount of any one
particular cDNA in the sample can be quantified by quantitative real-time PCR
using gene-specific primers.
random hexanucleotides means that all RNA molecules should be reversetranscribed
into cDNA. For the subsequent QRT–PCR reaction genespecific
primers are used. These primers direct the amplification of a short
segment of the cDNA. How QRT–PCR is used to quantify the amount of
DNA, in this case cDNA, is described in Section 3.18.
Q11.4. The QRT–PCR protocol utilizes random hexanucleotide primers to
generate the cDNA prior to QRT–PCR.What is the advantage of using random
hexanucleotides rather that a gene-specific primer?