Biomedical Engineering Reference
In-Depth Information
Method
Reverse transcription
1 Prepare an RT master mix of volume sufficient for the number of RNA samples being
analysed. For each RNA sample, prepare 18.75
μ
l of RT master mix by combining the
following:
•
12.75
μ
l of water
•
2.5
μ
lof10
×
RT buffer
•
2.5
μ
lof50ng/
μ
l oligo-dT
•
1
μ
lof4m
M
dNTP mix.
2 Make up two 'minus RT' controls for each RNA sample by combining 18.75
μ
lofmaster
mix with 6.25
μ
l of water.
3 Add 1.25
μ
l of RT to every 18.75
μ
l of RT master mix to give a final concentration of
2.5 U/
μ
l.
b
4 Add 20
μ
lofmastermixto5
μ
l of each RNA sample (preferably testing each sample in
duplicate), aiming for a target concentration of 50-500 ng/
μ
l (ensuring that each
reaction contains the same RNA concentration). Ensure that the final reaction volume
is 25
μ
l.
5 Incubate at 20
◦
C for 10 min followed by 50
◦
C for 60 min.
6 Terminate the reactions by heating to 85
◦
C for 5 min and place them on ice for 2 min,
or until required. Collect by brief centrifugation prior to continuing with step 10.
qPCR
7 For each single-stranded cDNA sample, prepare 20
μ
l of qPCR master mix by combining
the following:
•
12.5
μ
lof2
×
qPCR master mix buffer
•
4.5
μ
lof25m
M
MgCl
2
(4.5 m
M
final concentration)
•
0.25
μ
l of each 10
μ
M
primer (six primers)
•
0.5
μ
l of each 5
μ
M
probe (three probes).
8 Prepare a standard curve template as follows. Prepare seven 10-fold serial dilutions of
target-specific amplicon in yeast tRNA (100 ng/
μ
l) using a range of approximately 10
7
to 10
1
copies. Alternatively, use six fivefold dilutions of cDNA (where the highest
concentration is 1 : 2 dilution of cDNA synthesis) prepared from universal RNA, or
sample RNA. In each case, carry out the reactions in duplicate.
9Add5
μ
l of standard curve template to the appropriate qPCR reaction tubes, or
microtiter plate wells.
10 Prepare 1 : 10 dilutions (in water) of the cDNA from step 6 and add 5
μ
l of each diluted
sample to two qPCR reaction tubes.
