Cell Biology JoVE (Open Access) Citable · DOI

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

Name: In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
Uploaded: 2026-06-17
Duration: 8 min 55 s
Description: In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

DOI: 10.3791/3702-v

What you'll learn

✓ Synthesize high-yield capped and uncapped mRNA in vitro using T7 polymerase
✓ Enzymatically cap mRNA using Vaccinia virus capping enzyme
✓ Transfect mRNA into HeLa cells and measure luciferase reporter activity
✓ Optimize mRNA modifications for improved protein expression and detection

Protocol

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Difficulty

intermediate

Total time

~4–6 hours (includes in vitro transcription, capping, cell transfection, and assay incubation)

Model organism

HeLa

Biosafety

BSL-1

Steps

Synthesize Gaussia luciferase mRNA via in vitro transcription

Use the T7 High-yield RNA Synthesis Kit to generate capped and uncapped mRNA from linearized plasmid. Perform transcription reaction at appropriate temperature and duration to maximize RNA yield.

▶ 01:33

Cap uncapped mRNA using Vaccinia capping enzyme

Treat a fraction of purified uncapped RNA with Vaccinia virus capping system to enzymatically add 7-methylguanosine cap. This step improves mRNA stability and translation efficiency.

▶ 03:29

Transfect capped mRNA into HeLa cells

Use the TransIT mRNA Transfection Kit to deliver capped and uncapped mRNA into cultured HeLa cells according to manufacturer protocol. Optimize transfection conditions for maximum cell viability and transgene expression.

▶ 04:17

Measure Gaussia and Cypridina luciferase activity

Collect culture supernatants from transfected HeLa cells and perform luciferase assays using Gaussia and Cypridina luciferase substrates. Quantify reporter activity to assess mRNA translation efficiency.

▶ 06:08

Interpret representative results and data analysis

Evaluate luciferase activity differences between capped versus uncapped mRNA treatments. Compare knockdown or expression profiles to validate mRNA capping effects on protein production.

▶ 07:20

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