Biology Reference
In-Depth Information
3.2 Reverse
Transcribing mRNA
into cDNA
Using reverse transcription, mRNA produces a single-stranded
complementary DNA (cDNA) sequence. The cDNA is not present
in the cell under physiologic conditions, is an accurate refl ection of
the mRNA, and is easier to examine. To prepare a cDNA, a pd(N)6
primer is bound to one end of the mRNA molecule. This double-
stranded area is the starting point for the reverse transcriptase
enzyme, which can produce a single-stranded DNA copy. The
cDNA chain is extended in the 5
direction, and the deoxyribo-
nucleotides are added using the RNA sequence as a template. The
result is a hybrid molecule consisting of the mRNA template strand
and the complementary DNA strand, which is bound through
Watson-and-Crick base pairing. The single-stranded cDNA can be
separated from the RNA strand using heat. This cDNA can be
amplifi ed further using the PCR, which produces a large quantity
of the DNA sequence that represents the mRNA.
′
-3
′
1. To remove any genomic DNA residues, the total RNA is
treated with DNase I for a second time before it is rewritten
into cDNA (
see
Note 7
). The DNase I reaction is performed as
follows: 10
μ
l RNA solution, 1.5
μ
l 10× RT buffer, 3.3
μ
l
l
DNase I are placed into 0.5-ml Eppendorf tubes, which are
vortexed thoroughly.
2. The mixture is incubated in a thermoblock (Biometra,
Germany) for 20 min at 37 °C, followed by 8 min at 75 °C,
and the mixture is cooled briefl y on ice.
3. The total RNA is transcribed into cDNA using the TaqMan
®
Reverse Transcription Reagent Kit (Applied Biosystems, USA)
as follows: 15
MgCl2 (25 mM), 0.5
μ
l RNase inhibitor (20 U/μl),
μ
l), and 0.3
μ
μ
l of the DNase I-treated RNA solution is added
to 1.5
μ
l 10× RT buffer, 3.3
μ
l MgCl
2
(25 mM), 6.0
μ
l dNTPs,
1.5
μ
l random hexamer primers (5 nM), 0.5
μ
l RNase inhibitor
(20 U/
μ
l), and 0.8
μ
l MultiScribe™ reverse transcriptase
(50 U/
μ
fi mixed thoroughly, and diluted to a fi nal volume of
l using nuclease-free water.
4. The sample is incubated for 10 min at 25 °C, 45 min at 48 °C,
and 5 min at 95 °C using a Trio-Thermoblock (Biometra).
5. The prepared cDNA solution is stored at −20 °C until further
processing.
30
μ
Developed by Mullis and Fallona in 1987, the polymerase chain
reaction (PCR) can selectively multiply desired target genome seg-
ments. PCR uses DNA polymerases that can polymerize a single
DNA strand into a double strand provided that a short double-
stranded region is available as a primer. The fi rst step is to denature
the double-stranded DNA into two single strands by heating to
95 °C, which enables the binding of the primer. Next, the single
strands are hybridized using two sequence-specifi c oligonucleotides
(primers), which are complementary to the sequences on either side
of the target region. This step is termed the “annealing” step and
3.3 Real-Time
Polymerase Chain
Reaction
