Both methods are good but western blot has an edge!

Hello Rachana L. Patnayak

It is always better to confirm knockdown of your target at mRNA level. The reason being that siRNA targets mRNA for degradation. Therefore, qPCR is the most direct detection method to measure siRNA-mediated target knockdown. Also, qPCR is quantitative. This makes troubleshooting and optimizing experimental conditions much less complex than protein-based data.

If I were you, I would analyze knockdown with multiple assays and on multiple levels. Once a reasonable knockdown of your mRNA is detected, you can further optimize time courses to follow up on protein production or phenotypic changes. Western Blot could be used as follow-up experiment.

Best.

Hi Rachana L. Patnayak

RT-qPCR is commonly used to detect mRNA levels, but when siRNA is employed, it can result in stochastic outcomes with low reproducibility; therefore, a primer specificity test should be conducted beforehand. It is advisable to combine both methods, using different time frames. For siRNA experiments, the optimal time frames range from 24 to 48 hours, allowing for repeated transfections. Specifically, the experimental timeline would involve cell seeding, followed by siRNA transfection after 24 hours, a subsequent 36-hour waiting period, another round of transfection, an additional 36-hour waiting period, and finally, collection of mRNA and proteins for analysis

Both qPCR (quantitative polymerase chain reaction) and western blotting are commonly used techniques to assess gene knockdown in hybridoma cells. However, the choice between the two methods depends on several factors and the specific requirements of your experiment.

The choice between qPCR and western blotting depends on the nature of your research question, the specific gene you are targeting, and the available resources. It is often recommended to use both techniques in combination to obtain a comprehensive assessment of gene knockdown. qPCR can provide insights into changes at the mRNA level, while western blotting can confirm the corresponding protein-level effects.

Additionally, it is important to consider the time course of gene knockdown. qPCR can detect changes in mRNA levels relatively quickly, within hours or days of knockdown, whereas changes in protein levels may take longer to manifest. Therefore, if you are interested in early time points, qPCR may be more suitable, while western blotting could be employed for later time points to evaluate the protein reduction.

Malcolm Nobre Thanks for your answer. May I know, if you do total RNA isolation for qPCR or mRNA isolation?

Rachana L. Patnayak I perform total RNA isolation for qPCR.