Author: Joe Grove/Peter Balfe
HCV RNA extraction.
1. Out of cat 3; put 75ul protease (in box of mixed reagents in fridge), and
450ul DMEM into a 2ml tube.
2. Make up qiagen lysis buffer including carrier RNA (in freezer) using
protocol from book.
3. In cat 3 take 50ul of virus supernatant, add to 2ml tube containing
DMEM and protease. Then add 500ul lysis buffer and incubate at 56C
for 15-20 minutes to deactivate virus.
4. Remove virus prep from cat 3 (wiping tube with trigene) and follow
qiagen extraction protocol (from RNeasy protocol).
RTPCR Protocol.
1. Use basic set up as outlined in plate map (see below). Invitrogen kit
“cells direct” in PCR set up freezer, box should also contain GAPDH
primers. HCV primers are in a plastic box (as are spare GAPDH
primers), HCV primers are provided as powder, if starting new
resuspend in 300ul water. Orientate plate with pen in the left hand
corner.
2. Aliquot 10-15ul of RNA preps in to 8 well strips and use multi-channel
to pipette 2-3ul into the 96 well plate (perform in
triplicate/quadruplicate). HCV +ve RNA is in eppendorf with single
diagonal line on it (@109 copies/0.5ul).
3. Use larger multi-channel to pipette 12-15ul of master mix into each
well.
4. Cover plate with thermo-labile optically clear plastic and heat transfer
pad (do not loose latter).
5. Using new stratagene PCR machine; lift shutter and plate cover, make
sure plate is in right orientation.
6. Open program, quantitative PCR mode, open old template, make sure
its set for standard, vic and fam, alter thermal profile to one on plate
map, run, set a new file, check ‘turn bulb off after run’, start.
7. Run should take ~2.5hrs.
8. For analysis change standard curve wells from ‘unknown’ well type to
standards, designate copy numbers per well and which order they go
in, examine standard curve. If curve is looses shape at very low copy
numbers (i.e. 10-1000) take these out of the standard curve. The
program will then be able to give copy numbers for each sample which
can be exported as text report.
Example HCV RT-PCR Template.
17 samples.
RT-PCR Mix (100 wells):
750 µl 2 x PCR mix (Invitrogen, “CellsDirect” kit).
500 µl dH2O
45 µl GAPDH referent primer
45 µl HCV primer mix
30 µl RT-Taq enzyme mix
0.25 µl Hela RNA (Invitrogen) – required when using cell supernatants.
Split into 8 x 165 µl lots in 8 well strip.
Make up dilution series of control RNA in 8 well strip.
Add 2 µl RNA to each well as shown
Add 13 µl PCR mix across wells using multichannel
Plate map for date here, filename “file name”
1
2
3
4
5
6
7
8
9
10
11
12
A
1
9
108
108
108
108
B
2
10
107
107
107
107
C 3
11
106
106
106
106
D 4
12
105
105
105
105
E
5
13
104
104
104
104
F
6
14
103
103
103
103
G 7
15
102
102
102
102
H 8
16
PCR cycling: 30’ @ 50°C, 5’ @95°C. 50 x (95°C, 15s; 60°C, 60s).