siRNA / RNAi /miRNA transfection Human - Jurkat cells Lipofectamine

The RNA interference (RNAi) is used to inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Two types of RNA molecules such as microRNA (miRNA) and small interfering RNA (siRNA) play a central role in RNAi. Few points have to considered to increase the transfection efficiency of siRNA. Always use healthy, actively dividing cells to maximize transfection efficiency. The confluency of cells should be between 50-70%. Always use the most appropriate siRNA concentration to avoid off-target effects and unwanted toxic side effects. Positive and negative controls should be used for each and every experiment to determine transfection efficiency.

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1 year ago

1 year ago by Keith L. Morrison Canada

siRNA/RNAi/miRNA transfection human

I would like to regulate the expression of a gene and in order to do that, I have purchased specific siRNA. After optimizing my transfection protocol and using electroporation I have achieved a 60-70% reduction of the gene of interest. However, I cannot observe a significant reduction of mRNA expression but only a reduction of protein. What might be the problem? Could the problem be in my cell treatment method?

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Protocol tips
siRNA sequence:sequences are 5′-CAUCUCCUAUGGCGGUGGU (siNB1i) and 5′-UGGUCCUCCUCAAGGCAGU (siNB2i)

Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). 

Incubate for 5 minutes at room temperature and add to cells. 

Incubate cells for 3 days at 37°C. 
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