Biomedical Engineering Reference
In-Depth Information
PROTOCOL 6.4 Reverse Transcription Using
Oligo-dT Priming
Equipment and reagents
•
RNA (10-500 ng)
m
•
Oligo-dT (500 ng/
μ
l)
•
Reverse transcriptase (RT) 200 U/
μ
l
n
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
l of 500 ng/
μ
l oligo-dT
•
0-9.0
μ
l of water (to adjust the total volume to 10
μ
l).
2 Incubate at 65
◦
C for 10min, and snap cool on ice for 5 min.
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
o
•
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.
n
Gently mix the tube contents and briefly centrifuge.
5 Incubate at 20
◦
C for 10min followed by 50
◦
C for 60 min.
6 Terminate the reaction by incubating at 85
◦
C for 5 min, and then place on ice for 5 min.
Collect the reaction mixtures by brief centrifugation.
p
Notes
m
Each reaction must contain the same final concentration of RNA (maximum 20 ng/
μ
l). Hence
the most dilute sample will determine the total concentration used for all reactions.
n
Each reaction must contain the same final concentration of RNA (maximum 20 ng/
μ
l). Hence
the most dilute sample will determine the total concentration used for all reactions.
o
Ensure that manufacturers recommendations for specific enzymes are consulted.
p
First strand cDNAs can be stored at
−
20
◦
C for at least six months.
