QIAGEN Viral RNA UM Kit User Manual

Sample to Insight__

October 2020

Quick-Start Protocol

QIAprep& Viral RNA UM Kit

The QIAprep& Viral RNA UM Kit (cat. nos. 221415 and 221417) should be stored
immediately upon receipt at –30 to –15°C in a constant-temperature freezer and protected
from light. The Viral RNA Master Mix and ROX Reference Dye can also be stored at 2–8°C
for up to 12 months protected from light, depending on the expiration date.

Further information



QIAprep& Viral RNA UM Kit Handbook

 Safety Data Sheets: www.qiagen.com/safety

 Technical assistance: support.qiagen.com

Notes before starting

 The QIAprep& Viral RNA UM Kit is an innovative liquid-based method optimized for

the preparation and detection of viral RNA targets from samples such as nasal,
nasopharyngeal, or oropharyngeal swabs that are stored in non-fixation transport media
such as UTM, VTM, PBS, ESwabs

 Samples can be kept at room temperature during preparation steps and reaction setup.

Sample preparation can conveniently be performed directly in the PCR vessel prior to the
addition of the PCR reaction.

 The Viral RNA UM Prep Buffer prepares the samples for the detection step but is not a

virus inactivation solution.

 The RT-qPCR protocol uses TaqMan probes in a multiplex reaction that works with any real-time

cyclers. For fluorescence normalization, ROX dye might be required at the following concentrations:

 Low concentration of ROX dye: Applied Biosystems 7500, ViiA7, and QuantStudio

Real-Time PCR Systems.

 High concentration of ROX dye: ABI PRISM 7000, Applied Biosystems 7300, 7900,

and StepOne Real-Time PCR Systems.

 No requirement for ROX dye: Rotor-Gene, QIAquant, Bio-Rad CFX,

Roche LightCycler 480, and Agilent Technologies Mx instruments. The

®

, Virocult™, or 0.9% NaCl.

: www.qiagen.com/HB-2830

2

QuantiNova ROX Reference Dye should be used as a 20x concentrated solution for a
1x reaction when using an instrument requiring a high-ROX dye concentration. For
instruments requiring a low-ROX dye concentration, use the dye as a 200x concentrate.

 Important: Always start with the cycling conditions specified in this protocol.
 The PCR section of the RT-qPCR protocol must start with an initial incubation step of 2 min

at 95°C to activate the DNA Polymerase.

 The RNA IC Template + Assay (Internal Control) is an inhibition control using a synthetic

RNA template. It is a 200 bp IC template detected in the red channel on the

®

Rotor-Gene Q or in the Cy5

 The Human Sampling IC Assay (Sampling Control) is intended to report that the primary

channel on other real-time PCR instruments.

sample tube contains intact human genetic material. For this purpose, two different
human targets are both detected in the yellow channel on the Rotor-Gene Q or in the

®

/HEX dye channel on other real-time PCR instruments. The pre-mixed formulation

VIC
(20x) contains forward and reverse primers and TaqMan probes.

 For viral targets, it is recommended to prepare a 20x primer–probe mix containing

target-specific primers and probes. Viral sequences can be detected in the green channel
on the Rotor-Gene Q or in the FAM dye channel on other real-time PCR instruments. We
recommend to use 0.8 µM primers (forward/reverse) and 0.25 µM probe concentrations
in the reaction. For further information, or to download the handbook or supplementary
protocols, please visit the product page (www.qiagen.com/qiaprepandamp-resources).

 Before use, thaw the Viral RNA UM Prep Buffer, Viral RNA Master Mix, RNA IC

Template + Assay, Human Sampling IC Assay, ROX Reference Dye (if required), and
RNase-Free Water. Mix the individual solutions.

Procedure

1. Prepare a reaction mix according to Table 1 and mix thoroughly.

2. Vortex the swab containing the sample vigorously.

3. Dispense 2 µl of Viral RNA UM Prep Buffer into each PCR tube or a well of a PCR plate.

4. Add 8 µl of the sample to the same PCR tube or well containing the Viral RNA UM Prep
Buffer. Mix by pipetting up and down at least twice.

5. Incubate at room temperature for 2 min.

Note: Incubation time starts after adding the last sample to the Viral RNA UM Prep Buffer.

6. Add 10 µl of the reaction mix prepared in step 1.

QIAprep& Viral RNA UM Kit 10/2020