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RNAi - RNA Interference
Overview
RNA interference is a natural process that cells use to regulate gene expression. It involves small RNA molecules, such as small interfering RNA or micro RNA that can specifically target and degrade messenger RNA molecules that encode a particular gene. MRNA is a molecule that carries genetic information from DNA to the ribosomes, where it is translated into a protein. SiRNA is then introduced into the cell and binds to the protein complex called the RISC. The RISC then uses the small RNA as a guide to locate and bind to the complementary mRNA molecule that encodes the targeted gene.
Required materials
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siRNA molecules
-
Transfection reagent
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Cell culture media
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Target cells
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Opti-MEM media agent
Protocol
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Determine the sequence of the mRNA you wish to target with the siRNA and design siRNA duplexes to target the gene using software or online tools.
-
Reconstitute the siRNA duplexes in RNase-free water to a concentration of 20uM.
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Dilute the siRNA duplexes in Opti-MEM media to a final concentration of 20uM.
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In a separate tube, dilute the transfection reagent in the Opti-MEM media to a concentration of 0.1 μl per well
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Add Dilute siRNA Duplexes to the diluted transfection reagent and mix gently
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Incubate the mixture at room temperature for 20 minutes to allow the siRNA-lipid complexes to form.
-
While the complexes are forming, prepare cells for transfection by seeding them into the culture plates and allowing them to attach and grow overnight to achieve 50-80% confluency.
-
Add the siRNA-lipid complexes to the cells using 50-100nM of siRNA per well. Mix gently by rocking the plate back and forth.
-
Incubate the cells with the siRNA-lipid complexes at 37C for 24-72 hours, depending on the specific experiment.
-
After incubation, remove the media containing the siRNA-lipid complexes and replace them with fresh culture media.
-
Harvest the cells for analysis at appropriate time points, and assess gene knockdown using qPCR, Western blotting, or immunofluorescence techniques.
Results
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The expected results of RNA interference can vary as well as be many different forms of analysis, here are some common types of results that can be obtained from RNA interference experiments.
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Phenotypic changes: This is the most common type of result obtained from RNAi Experiments. This change can be observed in cells or organisms after the gene silencing, this may include changes in cell morphology, cell viability, proliferation, differentiation, apoptosis, migration, invasion, or specific cellular processes.
-
Knockdown efficiency: The level of knockdown of the targeted gene can be measured using techniques such as quantitative PCR or western blotting.
-
Changes in gene expression: Genes can be analyzed by techniques such as mirror analysis, these results can provide insight into the biological pathways regulated by the target gene.
siRNA molecules
Transfection reagent
Cell culture media
Target cells
Opti-MEM media agent
-
Determine the sequence of the mRNA you wish to target with the siRNA and design siRNA duplexes to target the gene using software or online tools.
-
Reconstitute the siRNA duplexes in RNase-free water to a concentration of 20uM.
-
Dilute the siRNA duplexes in Opti-MEM media to a final concentration of 20uM.
-
In a separate tube, dilute the transfection reagent in the Opti-MEM media to a concentration of 0.1 μl per well
-
Add Dilute siRNA Duplexes to the diluted transfection reagent and mix gently
-
Incubate the mixture at room temperature for 20 minutes to allow the siRNA-lipid complexes to form.
-
While the complexes are forming, prepare cells for transfection by seeding them into the culture plates and allowing them to attach and grow overnight to achieve 50-80% confluency.
-
Add the siRNA-lipid complexes to the cells using 50-100nM of siRNA per well. Mix gently by rocking the plate back and forth.
-
Incubate the cells with the siRNA-lipid complexes at 37C for 24-72 hours, depending on the specific experiment.
-
After incubation, remove the media containing the siRNA-lipid complexes and replace them with fresh culture media.
-
Harvest the cells for analysis at appropriate time points, and assess gene knockdown using qPCR, Western blotting, or immunofluorescence techniques.
Results
-
The expected results of RNA interference can vary as well as be many different forms of analysis, here are some common types of results that can be obtained from RNA interference experiments.
-
Phenotypic changes: This is the most common type of result obtained from RNAi Experiments. This change can be observed in cells or organisms after the gene silencing, this may include changes in cell morphology, cell viability, proliferation, differentiation, apoptosis, migration, invasion, or specific cellular processes.
-
Knockdown efficiency: The level of knockdown of the targeted gene can be measured using techniques such as quantitative PCR or western blotting.
-
Changes in gene expression: Genes can be analyzed by techniques such as mirror analysis, these results can provide insight into the biological pathways regulated by the target gene.
The expected results of RNA interference can vary as well as be many different forms of analysis, here are some common types of results that can be obtained from RNA interference experiments.
Phenotypic changes: This is the most common type of result obtained from RNAi Experiments. This change can be observed in cells or organisms after the gene silencing, this may include changes in cell morphology, cell viability, proliferation, differentiation, apoptosis, migration, invasion, or specific cellular processes.
Knockdown efficiency: The level of knockdown of the targeted gene can be measured using techniques such as quantitative PCR or western blotting.
Changes in gene expression: Genes can be analyzed by techniques such as mirror analysis, these results can provide insight into the biological pathways regulated by the target gene.