Protocol for Studying RSV Transcription using Minigenome Systems

Experiment Summary

Minigenome assays have been essential tools in the understanding of viral transcription and RNA replication for respiratory syncytial virus (RSV). Here, we describe the RSV minigenome assay for determining transcription by the viral polymerase in the absence of infection. We detail two different methods of detecting viral RNA synthesis: a firefly luciferase assay for rapid and sensitive measurement of RSV polymerase activity; and a real-time quantitative PCR method for determination of specific effects on the transcription of individual viral genes and the polar transcription gradient of RSV.

Materials and Reagents

A. Transfection

BSR-T7/5 cells.
Dulbecco's minimal essential medium (DMEM). Store at 4°C.
Complete Medium (CM): DMEM containing 10 % (v/v) fetal bovine serumand (FBS) 1× MEM nonessential amino acids. Store at 4°C.
Transfection reagent. Store at 4°C.
Plasmids: pcDNA3.1-N, pcDNA3.1-P, pcDNA3.1-M2-1, pcDNA3.1-L; C2L and D53; phRL-TK. Store at -20°C.
24-well plates.

B.Luciferase Assay

Dual luciferasereporter assay kit. Store at -80°C.
Phosphate buffered saline (PBS): 10× (80 g NaCl, 2 g KCl, 11.5 g Na₂HPO₄⋅7H₂O, 2 g KH₂PO₄ in 1 L ultrapure water before use). Dilute to 1× in ultrapure water before use.
Black flat-bottom 96-well plate.
Plate reading luminometer.
Distilled water.
70 % ethanol in distilled water.

C. Real-Time Quantitative PCR (qPCR) Assay

RNAzol.
Phosphate buffered saline(PBS): 10× (80 g NaCl, 2 g KCl, 11.5 g Na₂HPO₄⋅7H₂O, 2 g KH₂PO₄ in 1 L ultrapure water). Dilute to 1× in ultrapure water before use.
Isopropanol.
Nuclease-free water.
75 % ethanol in nuclease-free water.
cDNA synthesis kit. Store at -20 °C.
2× SYBR Green qPCR kit. Store at −20 °C.
Primers.
96-well PCR plate, 0.2 ml well.
Optical Film PCR plate cover.
Standard PCR machine.
Real-time PCR machine.

Procedure

A. Transfection

Plate BSR-T7/5 cellsin 24-well clusters (~2 × 10 4 cells/well) in complete medium (CM). Seed enough wells for triplicate samples.
Incubate overnight at 37°C/5 % CO₂.
Mix support plasmids in a sterile 1.5 ml microfuge tube.
Make a master mix of transfection reagent (3 μl GeneJuice/μg DNA) in serum -free DMEM (250 μl/tube). Incubate for 5 min.
Add 250 μl of transfection reagent master mix to each tube, mix by gentle agitation, and incubate for 30 min.
Remove media from 24-well plate of BSR-T7/5 cells.
Add 83 μl of DNA/GeneJuice mix per well and incubate at 37 °C/5 % CO₂ for 24 h.
Harvest cells for luciferase or qPCR assay.

B. Luciferase Assay (Dual Luciferase Reporter Assay System Method)

Dilute the 5× Passive Lysis Buffer (PLB) in distilled water to make a 1× solution. Make sure PLB is at room temperature before use.
Remove media from cells and wash with 1× PBS by rocking plate gently to remove detached cells and residual medium.
Completely remove PBS before adding PLB.
Add 100 μl PLB to each well (24-well plate), swirl to cover cells. Rock plate for 30 min. Transfer lysate to 0.5 ml microfuge tube and keep on ice.
Spin samples at max speed (13,000 × g) for 1 min to clarify.
Prepare Luciferase Assay Reagent II (LAR II) by solubilizing the lyophilized Luciferase Assay Substrate in 10 ml of the Luciferase Assay Buffer II.
Preparation of Stop & Glo Reagent.
Program plate reader luminometer to perform a 2-s premeasurement delay, followed by a 10 s measurement period for each reporter assay. Program the injector system to dispense 50 μl of diluted LARII reagent per well and 50 μl of diluted Stop & Glo reagent per well.
Purge all storage liquid (distilled water or 70 % ethanol wash solution) from injector. Prime empty injector system with LAR II (Injector #1) or Stop & Glo Reagent (Injector #2) and collect priming reagent in appropriate vials.
Dispense 30 μl (for most luc assays from p24, I usually use 30 μl) of cell lysate samples into appropriate well of black 96-well microplate. Place the microplate in plate reader.
Run Dual Luciferase Assay program. Make sure injection volume is set to 50 μl as the Synergy Mx will inject 100 μl otherwise.
Download the data from the Excel file. Calculate firefly luciferase numbers relative to Renilla luciferase.

C. General Injector Wash Protocol

Purge Stop & Glo Reagent from injector lines by repeated priming/washing with distilled water.
Prime injector system with at least 5 ml 70 % ethanol to completely replace the void volume and rinse the injector plumbing. It is preferable to soak in this wash solution for 30 min prior to rinsing with distilled water.
Rinse with enough distilled water (at least 3 pump void volumes) to thoroughly remove all traces of ethanol.

D. RNA Isolation

Aspirate medium from wells of transfected cells.
Wash cells with 1 ml ice-cold PBS and aspirate.
Add 250 μl of RNAzol and incubate for 5 min at room temperature.
Harvest the RNAzol mixture by carefully swirling the viscous liquid around the well and add to a 1.5 ml microfuge tube.
Add 100 μl nuclease-free water to each tube, mix by inversion 6-10 times, and incubate for 15 min.
Centrifuge tubes for 10 min at 12,000 × g.
Transfer supernatant to fresh microfuge tube, add 1 vol. of isopropanol and mix by inversion. Incubate for 15 min.
Centrifuge at 12,000 × g, for 10 min.
Aspirate supernatant, add 250 μl 75 % ethanol, and centrifuge at 4000 × g, for 2 min.
Repeat step 9.
Resuspend pellet in 10 μl of nuclease-free H₂O.

E. First Strand cDNA Synthesis

F. Real-Time Quantitative PCR Assay

* For research use only. Not intended for any clinical use.

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Protocol for Studying RSV Transcription using Minigenome Systems

Experiment Summary

Materials and Reagents

Procedure

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