Real time RT-PCR on HCV RNA isolated from the cell culture supernatant and plasma.
Part 1: isolation of HCV RNA.
HCV RNA can be extracted from cell culture supernatant, plasma or serum using the High Pure Viral
Nucleic Acid Kit (Roche). The RNA will be eluted by 50 µl Rnase free water (Supplied with the kit).
Procedure:
1. All solutions provided with the kit should be clear. If precipitations are formed then warm the
solutions in a 37°C waterbath until the precipitations have dissolved.
2. Preparation of working solutions:
a. Dissolve the Proteinase K (100 mg, vial 3, pink cap) in 5 ml Elution Buffer (vial 5)
and mix thoroughly by inversion (do not vortex). Prepare 1 ml aliquots, store at -20°C
(stable for 12 months).
b. Reconstitute the lyophilized Poly(A) carrier RNA (vial 2) in 0.5 ml Elution Buffer
(vial 5). Prepare 50 µl aliquots and store them at -20°C (stable for 12 months).
c. Thaw one 50 µl vial of carrier RNA and mix thoroughly with 2.5 ml of Binding
Buffer (vial 1). Prepare always fresh before use, do not store!
d. Inhibitor Removal Buffer (vial 4a, black cap): add 20 ml of absolute ethanol, mix
well. Label bottle and store at room temperature.
e. Wash buffer (vial 4, blue cap): add 40 ml of absolute ethanol, mix well. Label bottle
and store at room temperature.
3. Add to a nuclease-free 1.5 ml tube the following reagents:
a. 200 µl serum, plasma or cell culture supernatant. Samples that contain precipitates
must first be centrifuged!
b. 200 µl freshly prepared carrier RNA-supplemented Binding Buffer (step 2c)
c. 50 µl Proteinase K solution
Mix immediately and incubate for 10 min. at 72 °C
4. Add 100 µl Binding Buffer and mix.
5. Transfer content of vial (550 µl) to a High Filter tube, inserted in a collection tube. Centrifuge
for 1 min. at 8.000 g.
6. Remove filter tube from collection tube. Discard flow through liquid and the used collection
tube. Combine the filter tube with a new collection tube.
7. Add 500 µl Inhibitor Removal Buffer and centrifuge for 1 min. at 8.000 g.
8. Remove filter tube from collection tube. Discard flow through liquid and the used collection
tube. Combine the filter tube with a new collection tube.
9. Add 450 µl Wash buffer and centrifuge for 1 min. at 8.000 g.
10. Remove filter tube from collection tube. Discard flow through liquid and the used collection
tube. Combine the filter tube with a new collection tube.
11. Add 450 µl Wash buffer and centrifuge for 1 min. at 8.000 g.
12. Discard the flow through liquid and recombine the filter tube with the used collection tube.
Centrifuge for 10 sec. at 13.000 g to remove any residual Wash buffer.
13. Discard the collection tube and combine the filter tube with a nuclease-free, sterile 1.5 ml
microcentrifuge tube.
14. Add 50 µl of Elution Buffer and centrifuge for 1 min. at 8.000 g.
15. Discard the filter tube. The 1.5 ml microcentrifuge tube now contains the viral RNA: use
directly or store at -80°C for further analysis.
Overview:
Part 2: quantification of isolated viral RNA.
Real Time RT-PCR will be performed using the LightCycler®480 RNA Master Hydrolysis Probes kit
(Roche). Wear gloves!
1. Thaw all reagents from the kit and spin the vials briefly before opening. Mix carefully by
pipetting up and down. Store on ice.
2. Prepare a 10X solution in PCR grade water containing both PCR primers (10 µM) and the
hydrolysis probe (1 µM). Make 150 µl aliquots, store at -20°C and protect from light.
3. Prepare an RT-PCR mix in a nuclease-free 1.5 ml tube for the amount of reactions needed +
one additional reaction. For each 20 µl reaction mix the following reagents:
Component
Volume/reaction
PCR grade water (vial 3)
3.3 µl (2.3 µl)
10X primer/probe mix
2.0 µl
Final concentration
Primers:
(preheat for 1 min. at 95°C )
HCV1-F 0,5 µM
HCV1-F 5 µM
HCV1-R 1 µM
HCV1-R 10 µM
Probe: 0.250 µM
Probe: 2,50 µM
Activator (50 mM ; green cap)
1.3 µl
3.25 mM
LightCycler 480 RNA Master 7.4 µl
Hydrolysis probes (2,7X; vial 1,
red cap)
1X
Enhancer (20X ; tellow cap)
1.0 µl
1X
Uracyl-DNA Glycosylase (10X) (1 µl)
Optional
1X
Total volume
15 µl
4. Mix carefully by pipetting up and down. Do not vortex!
5. Pipet 15 µl RT-PCR mix into each well of the LighCycler 96-well plate. Do not touch the
surface of the LightCycler 480 Multiwell Plate when handling it!
6. add 5µl of each sample, control or standard to each well
7. Seal the multiwell plate with LightCycler 480 Sealing Foil.
8. Place the multiwell plate in a centrifuge and centrifuge for 2 min. at 1500 g (3000 rpm).
9. Incubate multiwell plate for 5 min. at room temperature to destroy contaminating DNA
(optional).
10. Load the plate into the LightCycler 480 and start program “HCV RNA quantification”.
General remarks:

All samples, controls and standards should be run in triplicate.

100 % limit of detection:

Limit of quantification:

Linear range:

LyghtCycler 480 Program Name: “HCV RNA quantification”
Detection format
Block type
Reaction volume
Mono color hydrolysis probes
96 well
20 µl
Program name
Cycles
Analysis mode
Reverse transcription
1
None
Denaturation
1
None
Amplification
55
Quantification
Cooling
1
None
Hold (hh:mm:ss)
Ramp Rate (°C/s)
None
00:03:00
4.4
None
00:00:30
4.4
95
None
00:00:15
4.4
59
None
00:00:50
2.2
72
Single
00:00:01
4.4
None
00:00:10
2.2
Programs
Temperature Targets
Target (°C)
Acquisition mode
Reverse transcription
63
Denaturation
95
Amplification
Cooling
40
Materials and reagents needed.
Description
Company (Cat. N°)
Price
High Pure Viral Nucleic Acid Kit (100 isolations)
Roche (11 858 874 001)
229.50
Spare collection tubes
Yvonne/Sofie
1.5 ml nuclease-free tubes
Yvonne
Absolute ethanol
VWR (xxx)
PCR grade water
Lonza (xxx)
LightCycler 480 RNA Master Hydrolysis probes (5x 100 Roche (04 991 885 001)
reactions)
495.00
LightCycler 480 Multiwell plat 96, clear (5 x 10 plates incl. Roche (05 102 413 001)
sealing foil)
LightCycler 480 Multiwell plat 96, white (5 x 10 plates Roche (04 729 692 001)
incl. sealing foil)
227.50
LightCycler 480 Sealing Foil (50 foils)
Roche (04 729 757 001)
78.75
Uracyl-DNA Glycosylase (100 U)
Roche (11 775 367 001)
115.74
Uracyl-DNA Glycosylase (500 U)
Roche (11 775 375 001)
429.84