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RNA Construct Stability and Homogeneity in Ribosomal Lysate - Experimental Study

This study explores the homogeneity and stability of various mRNA constructs in a ribosomal lysate environment. Constructs including 5’UTR control, SL-5’UTR control, AUG control, SL-AUG control, and more were incubated for different durations and analyzed using denaturing agarose gel electrophoresis. Analysis of 18S and 28S rRNA was performed to visualize mRNA constructs' behavior. The experiment was carried out with both standard mRNA and HIV-2 derived sequences to compare stability and homogeneity. Findings shed light on translational efficiency and construct performance in ribosomal lysate.

terence-shirley
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RNA Construct Stability and Homogeneity in Ribosomal Lysate - Experimental Study

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  1. SL-5’UTR control – 30’ SL-5’UTR control – 0’ SL-AUG control – 30’ SL-AUG control – 0’ 5’UTR control – 30’ 5’UTR control – 0’ AUG control – 30’ AUG control – 0’ 28S rRNA 16S rRNA S1: Homogeneity and stability of the mRNA translated: A - Homogeneity and stability of the mRNA constructs in the RRL. 0.05 µM of 32P labeled 5’UTR Control, SL-5’UTR Control, AUGControl, SL-AUG control were incubated for 0 or 30 minutes in 15 µL RRL under translational conditions. The reaction was stopped on ice in the presence of 1% SDS, phenol extracted, ethanol precipitated, and the RNAs analysed on a 1% denaturing agarose gel. The 18S and 28S rRNA were visualized by SyBr staining before autoradiography and are indicated on the left hand side of the figure.

  2. SL-HIV-2-5’UTR –30’ SL-HIV-2-AUG1 – 30’ SL-HIV-2-5’UTR – 0’ SL-HIV-2-AUG1 – 0’ HIV-2-5’UTR – 30’ HIV-2-5’UTR – 0’ HIV-2-AUG1 –30’ HIV-2-AUG1 – 0’ 28S rRNA 16S rRNA B - Homogeneity and stability of the mRNA constructs in the RRL. 0.05 µM of 32P labeled HIV-2-5’UTR, SL-HIV-2-5’UTR, HIV-2-AUG1, SL-HIV-2-AUG1 were incubated for 0 or 30 minutes in 15 µL RRL under translational conditions. The reaction was stopped on ice in the presence of 1% SDS, phenol extracted, ethanol precipitated, and the RNAs analysed on a 1% denaturing agarose gel. The 18S and 28S rRNA were visualized by SyBr staining before autoradiography and are indicated on the left hand side of the figure.

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