Biomedical Engineering Reference
In-Depth Information
PROTOCOL 6.5 Reverse Transcription Using Target-Specific Primers
Equipment and reagents
•
RNA (1-200 ng)
q
•
Target-specific (antisense) primer (2
μ
M
)
•
Reverse transcriptase (RT) 200 U/
μ
l
•
10
×
RT buffer (supplied with RT)
25m
M
MgCl
2
•
100m
M
DTT.
•
Method
1 Briefly centrifuge the RNA and primers and combine to prepare the following
pre-reaction mixture:
•
1.0-9.0
μ
lofRNA
•
1.0
μ
lof2
μ
M
target-specific (anti-sense) primer
•
0-9.0
μ
l of water (to adjust the total volume to 10
μ
l).
2 Incubate at 65
◦
C for 10min, and then snap cool on ice for 5min.
3 Prepare an RT master mix combining, for each RNA sample:
•
2.5
μ
lof10
×
RT buffer
•
5.0
μ
lof25m
M
MgCl
2
•
2.5
μ
l of 100m
M
DTT
•
1.0
μ
l of 200 U/
μ
lRT
r
•
4.0
μ
l of water.
4 Add 15
μ
l of the RT master mix to the RNA/primer mix to make a total volume of 25
μ
l.
s
Gently mix the tube contents and briefly centrifuge.
5 Incubate at 50-65
◦
C for 5-15min.
6 Terminate the reaction by incubating at 85
◦
C for 5min, and then place on ice for 5min.
Collect the reaction mixtures by brief centrifugation.
t
Notes
q
It is preferable (but not essential) for each reaction to contain the same final concentration of
RNA (maximum 20 ng/
μ
l). Hence the most dilute sample may determine the total concentration
used for all reactions.
r
Ensure that manufacturers recommendations for specific enzymes are consulted.
s
Prepare no-RT controls in duplicate by adding 14
μ
l of master mix and 1
μ
l of water prior to
adding the RT enzyme to the master mix. Add this 'no enzyme' mixture to the primed RNA from
step 2.
t
First strand cDNAs can be stored at
−
20
◦
C for at least six months.
