Biomedical Engineering Reference
In-Depth Information
PROTOCOL 6.3 Reverse Transcription Using Random Primers
Equipment and reagents
•
RNA (10-500 ng)
i
•
Random primers (6-mer, 9-mer or 15-mer; 50 ng/
μ
l)
•
Reverse transcriptase (RT) 200 U/
μ
l
•
10
×
RT buffer (supplied with RT)
25m
M
MgCl
2
•
100m
M
dithiothreitol (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
lof50ng/
μ
l random primers
•
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
j
•
4.0
μ
l of water.
4 Add 15
μ
l of theRT master mix to the RNA/primer mix to make a total volume of 25
μ
l.
k
Gently mix the tube contents and briefly centrifuge.
5 Incubate at 20
◦
C for 10min followed by 50
◦
C for 60min.
6 Terminate the reactions by incubating at 85
◦
C for 5min, and then place on ice for
5 min. Collect the reaction mixtures by brief centrifugation.
l
Notes
i
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.
j
Ensure that manufacturers recommendations for specific enzymes are consulted.
k
Prepare no-RT controls in duplicate by adding 14
μ
lofmastermixand1
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.
l
First strand cDNAs can be stored at
−
20
◦
C for at least six months.
