“GROBE-2STEPPCR”
DECEMBER 10, 2003
2 STEP REAL-TIME PCR:
STEP 1 – REVERSE TRANSCRIPTASE REACTION
1.
2.
3.
4.
Determine RNA concentration
a. Dilute aliquot of sample 1:100
b. Measure absorbance at 260 and 280 nm
c. Multiply 260 nm absorbance by 4 to determine
concentration (ug/uL)
Calculate volume (uL) required for 1 ug RNA for each sample
Dilute appropriate volume (uL) of each sample to 19.25 uL
with DEPC water (“RT” set – use as both ACE2 RT and 18S
RT)
Dilute a second identical set of samples (“NRT” set – use
as 18S NRT) to 19.25 uL with DEPC water
5.
Select highest concentration sample; use for standard curve
a. Determine volume (uL) required for 2, 0.4, 0.08, and
0.016 ug
b. Dilute appropriate amount to 38.5 uL with DEPC water
6.
Create a no template control (“NTC”) with 19.25 uL DEPC
water (no sample)
7.
Create master mixes;
a. Standard Curve [1x] (for 100 uL RXN’s)
i.
RT Buffer (10x)
10
uL
ii.
MgCl2
22
uL
iii.
dNTP
20
uL
iv.
Random Hexamers
5
uL
v.
RNase Inhibitors
1
uL
vi.
MultiScribe
1.25 uL
b.
RT Samples and NTC [1x] (for 50 uL RXN’s)
i.
RT Buffer (10x)
5
uL
ii.
MgCl2
11
uL
iii.
dNTP
10
uL
iv.
Random Hexamers
2.5 uL
v.
RNase Inhibitors
1
uL
vi.
MultiScribe
1.25 uL
vii.
DEPC Water
--
c.
NRT Samples [1x] (for 50 uL RXN’s)
i.
RT Buffer (10x)
5
ii.
MgCl2
11
iii.
dNTP
10
iv.
Random Hexamers
2.5
v.
RNase Inhibitors
1
vi.
MultiScribe
-vii.
DEPC Water
8.75
1 / 3
uL
uL
uL
uL
uL
uL
“GROBE-2STEPPCR”
DECEMBER 10, 2003
8. Add 61.5 uL Standard Curve Master Mix to Standards
9. Add 30.75 uL RT Master Mix to RT samples and NTC
10. Add 30.75 uL NRT Master Mix to NRT samples
11. Run all tubes in thermocycler
a. 25ºC, 10 minutes
b. 48ºC, 30 minutes
c. 95ºC, 5 minutes
d. 4ºC, [Hold]
STEP 2 – REAL-TIME PCR
1. Create Master Mixes for ACE2 and 18S
a. ACE2 Master Mix [1x] (for 25 uL RXN’s)
i. Universal PCR Mix
12.5
uL
ii. Forward Primer (100 uM)
0.225 uL
iii. Reverse Primer (100 uM)
0.225 uL
iv. Probe (1:4 from stock)
0.25 uL
v. DEPC Water
1.8
uL
b. 18S Master Mix [1x] (for 25 uL RXN’s)
i. Universal PCR Mix
12.5
uL
ii. Forward Primer (100 uM)
0.225 uL
iii. Reverse Primer (100 uM)
0.225 uL
iv. Probe
0.156 uL
v. DEPC Water
1.8
uL
2.
Pipette 15 uL appropriate master mix into each well of 96
well plate
3.
4.
5.
6.
Dilute
Dilute
Dilute
Dilute
ACE2 RT Samples 1:10 in DEPC Water
Standard Curve samples 1:10,000 in DEPC Water
18S RT Samples 1:10,000 in DEPC Water
18S NRT Samples 1:10,000 in DEPC Water
7.
Add 10 uL of undiluted Standard Curve samples to wells (in
triplicate) “ACE2 Standard Curve”
8. Add 10 uL of 1:10 diluted ACE2 Samples to wells (in
triplicate) “ACE2 Samples”
9. Add 10 uL of 1:10,000 diluted Standard Curve samples to
wells (in triplicate) “RT Standard Curve”
10. Add 10 uL of 1:10,000 diluted RT samples to wells (in
triplicate) “RT Samples”
11. Add 10 uL of 1:10,000 diluted NRT samples to wells (in
triplicate) “NRT Samples”
12. Add 10 uL of 1:10 NTC Sample to wells (in triplicate)
“ACE2 NTC”
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“GROBE-2STEPPCR”
DECEMBER 10, 2003
13. Add 10 uL of 1:10,000 NTC Sample to wells (in triplicate)
“RT NTC”
14. Place clear plastic adhesive cover on 96 well plate, and
press down with brown plastic spreader (BE SURE TO NOT
TOUCH THE TAPE-LIKE COVER WITH BARE HANDS)
15. Spin plate for 4 minutes at 2,000-4,000 rpm in centrifuge
in cold room
16. Place plate in real-time PCR machine
17. Place insulating pad on top of plate
18. Close real-time PCR machine door
19. Configure plate layout and cycle parameters on real-time
PCR computer
a. 45 cycles
b. 25 uL sample size
c. Be sure to “save” file before beginning run
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