After live-cell imaging, total RNA from U2OS cells were extracted by using Monarch Total RNA Miniprep kit (New England Biolabs) and treated with TURBO DNase (Invitrogen). RT-qPCR was performed by using Luna Universal One-Step RT-qPCR reagents (New England Biolabs), following the manufacturer’s instructions. Primers targeting the MS2 stem-loop regions were used for quantifying repeat RNA expression levels, which were normalized by GAPDH expression levels.
Luna universal one step rt qpcr reagents
Manufactured by New England Biolabs
The Luna Universal One-Step RT-qPCR reagents are a set of reagents designed for reverse transcription and real-time quantitative PCR in a single reaction. The reagents include a reverse transcriptase enzyme, a DNA polymerase, and necessary buffers and components to perform both steps simultaneously.
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2 protocols using luna universal one step rt qpcr reagents
1
Quantifying Repeat RNA Expression
2
Quantifying Repeat RNA and DPR Expression
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Expression constructs for repeat RNAs or GFP-tagged codon-optimized DPRs were transfected in HEK293 cells or U-2 OS cells with Lipofectamine 2000 (Invitrogen) or FuGENE HD (Promega), respectively, according to the manufacturers’ instructions. 24−48 h after transfection, total RNA was extracted with TRIzol and treated with Turbo DNase (Invitrogen). RT-qPCR was performed on a CFX96 RT-PCR system (Bio-Rad) with Luna Universal One-Step RT-qPCR reagents (New England Biolabs). Fifty nanograms of total RNA and 250 nM primers (see Source Data file for primer sequences) were added in each reaction. Ct values were averaged across two technical replicates and normalized to GAPDH internal controls. Three independent replicate experiments were typically performed.
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