We see protein knockout by Western Blot analysis, but not by RT-PCR. How is possible?

Dear Esteemed Colleague,

Your observation regarding the discrepancy between Western Blot analysis and RT-PCR results in the context of protein knockout experiments is indeed intriguing and warrants a detailed examination. There are several plausible explanations for this phenomenon, each rooted in the fundamental differences between these methodologies and the biological processes they assess. Let us explore these explanations to provide a clearer understanding of the situation.

Post-transcriptional Regulation: One of the primary reasons for observing a protein knockout via Western Blot and not through RT-PCR is the presence of post-transcriptional modifications and regulatory mechanisms. While RT-PCR measures mRNA levels, indicating the gene's transcription, Western Blot analyzes the protein product. It is possible that the mRNA of the targeted gene is still being transcribed, but post-transcriptional regulation prevents the translation of this mRNA into protein. Mechanisms such as mRNA degradation, microRNA regulation, or inhibition of translation initiation factors could contribute to this discrepancy.

Protein Stability and Degradation: Even if the gene knockout does not completely stop the transcription of the target gene, changes in the protein's stability or an increase in its degradation rate could lead to the absence of the protein, as detected by Western Blot. Certain knockout mutations might affect protein folding or stability, leading to rapid degradation via proteasomal pathways, thus reducing the detectable protein levels despite the presence of its mRNA.

Sensitivity and Specificity of the Assays: The sensitivity and specificity of Western Blot and RT-PCR techniques might also play a role in this observation. Western Blot is highly specific to the protein of interest, provided that a specific and high-affinity antibody is used. In contrast, RT-PCR primers might not be specific enough or could amplify non-target sequences, leading to the detection of mRNA transcripts that do not result in the production of the functional protein. Additionally, the sensitivity of Western Blot in detecting low levels of protein may surpass the detection limits of RT-PCR for its mRNA, especially in cases of highly efficient translation or post-translational stability.

Alternative Splicing: The gene of interest might undergo alternative splicing, producing different mRNA isoforms. Some of these isoforms might not be targeted by the knockout strategy or the primers used for RT-PCR, allowing them to escape detection at the mRNA level but leading to a significant reduction or absence of the protein product detected by a Western Blot.

Technical Variabilities: Lastly, technical aspects and experimental conditions specific to each method could contribute to these differences. These include the efficiency of the RNA extraction method, the quality of the mRNA sample, the efficiency of cDNA synthesis, and the choice of primers for RT-PCR, as well as the choice of antibodies and detection sensitivity for Western Blot.

In summary, the discrepancy between Western Blot and RT-PCR results in protein knockout experiments can arise from a multitude of biological and technical factors. Understanding these nuances is crucial for interpreting experimental outcomes accurately. Further investigations, possibly including additional control experiments, validation of mRNA and protein sequences targeted, and examination of post-transcriptional regulatory mechanisms, could provide deeper insights into this observation.

Best regards,

Check out this protocol list; it might provide additional insights for resolving the issue.

Muhammad Saif Ur Rahman

Assuming your experiments are technically OK, we still can't assume mRNA as an indicator of gene regulation.

Certain post transnational modifications are key to final synthesis of the native proteins.

Sharmin Hasan

Hi Andrea,

Since, your KO is 70-80% successful, the rest other 30-20% RNA still remained which got multiplied by PCR reactions making millions of copies. Thus, RT-PCR could not show a significant difference with the WT vs KO. RT-PCR is usually used to detect temporal expression, not fully trustworthy to detect a 70-80% KO phenomenon. Is their any reliable antibody for the target gene? WB against that antibody could give a straight forward answer than RT-PCR.

Hope this helps. Best wishes!

Andrea Emanuelli

We tested the specificity of the antibody for WB and for me, as long as we see ko by wb, this is hopefully enough to continue my experiments. Also primers for rt-pcr look good and specific. The point is that in other cell lines I can easily see the knockout either by wb and rt-pcr although the ko is not 100%. For rt-pcr I used Viia7 system with SYBR green, and the Ct for that gene coming from wt and ko samples are the same. This means that both samples contain the same amount of mRNA for that gene, if I am correct. I just don't understand how this is possible. I thought Crispr-cas9 did not work, but I see the difference in wb analysis....

Ümit Uzun

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