Obtaining Viral RNA for Real-Time Quantitative Polymerase Chain Reaction (qPCR) Analysis

Abstract

Source: Suzuki, Y., et al. Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction. J. Vis. Exp. (2018).

This video demonstrates the procedure for preparing viral samples for quantitative PCR (Polymerase chain reaction) analysis. Viral samples and RNA standards are serially diluted, treated with a proteinase K buffer, and incubated under controlled temperatures to release RNA. The processed RNA is then stabilized and stored for quantification of viral RNA levels.

Protocol

1. Processing of Virus Samples for RT-qPCR (Real-time quantitative polymerase chain reaction)

NOTE: Steps 1.1–1.3 are to be performed inside a biological safety cabinet. In particular, handling of the culture supernatant containing an infectious virus must be conducted under a biosafety level 2 (or higher) environment.

1. Mix 199 μL of a processing buffer and 1 μL of nuclease-free proteinase K.
2. Serially dilute the synthesized DENV (dengue virus) 3’UTR (Untranslated region) RNA (Ribonucleic acid) 1:10 to obtain 5 x 10⁹ to 5 x 10³ copies/μL of RNA standard using cell culture medium (e.g., DMEM (Dulbecco's modified eagle medium) supplemented with 10% fetal bovine serum and antibiotics [DMEM/10% fetal bovine serum]) containing 40 units/mL RNase inhibitor.
3. Mix 5 μL of the culture supernatant of DENV-infected cells and the DENV 3’UTR RNA standard with 5 μL of processing buffer/proteinase K solution using 8-tube PCR (Polymerase chain reaction) strips or a 96-well PCR plate. As a non-template control (NTC), mix 5 μL of cell culture medium (i.e., no virus is included) with 5 μL of the processing buffer/proteinase K solution.
4. After a brief centrifugation, incubate the samples in a thermocycler using the following conditions: 1 cycle (of 25 °C for 10 min and 75 °C for 5 min).
NOTE: Processed samples can be kept at 4 °C if the real-time PCR analysis is done on the same day. For long-term storage, the samples should be stored at -80 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SimpliAmp Thermal Cycler	Thermo Fisher Scientific	A24811	Comparable thermal cycler instrument, but PCR cycle condition needs to be optimized.
Recombinant RNase Inhibitor	Takara Bio. Inc	2313A	40 units/μL
RNeasy Mini Kit	Qiagen	74104	Comparable RNA purification kit can be used.
Proteinase K	Nacalai Tesque	15679-06	>600 units/mL. Comparable reagent can be used.
MicroAmp Fast Optical 96-Well Reaction Plate, 0.1 mL	Thermo Fisher Scientific	4346907	Comparable plate or tube can be used dependent on the real-time PCR instrument, but PCR cycle condition needs to be optimized.