RNAi technology — a new perspective on life
Since September 2004, "Nature" published the first article on the results of RNA interference by American scientist Andrew Z. Fire and Craig C. Mello et al., The small RNA plays the important regulatory role in biological life activities. Every day there are articles on relation of the various small RNA and body development. And smart biotechnology companies are also at an amazing rate to actively use RNAi technology for drug screening, Cloud-Clone Corp. has been launched for the lentivirus and adenovirus expression system, The RNAi is one of the important sever of customer.
RNAi is a specific gene silencing technology, which is mediated by double-stranded RNA (dsRNA). RNAi blocks gene expression at the transcriptional level, post-transcriptional level, and translation level. And it is widely found in life activities from a variety of creatures.
The experimental flow chart of gene silencing is as follows:
Using the human CDHE gene as an example, we selected three RNAi target sites by biological software and the literature. Three shRNA skeletons of the target site were inserted into the pLKO.1 vector. Then we obtained the recombinant plasmids: pLKO.1-shCDHE-1, PLKO.1-shCDHE-2 and pLKO.1-shCDHE-3. After correcting with the sequencing, the correct recombinant plasmid was selected for transient transfection.
How to determine the efficiency of RNA interference?
At the mRNA level, we can use RT-PCR (Real-time, PCR) to detect, and the results are shown in figure 1.
RT-PCR results showed that sh-CDHE-2 can reduce the level of CDHE mRNA in both cells.
At the protein level, we can use Western-blot, ELISA, immunohistochemical method for detection, and the test results are shown in Figure 2.
RT-PCR results showed that sh-CDHE-1 and 2 in 293T cells have a certain effect. The WB results showed that the sh-CDHE-1 and 2 can down-regulate the expression of related proteins, which consistent with RT-PCR results.
The RNAi technology can specifically interfere the expression of specific genes (the length of more than thirty dsRNA can cause interferon toxicity). So the technology has been widely used to explore gene function, infectious diseases and malignant tumors in the field of treatment.
Cloud-Clone Corp is further tapping the potentially powerful features of RNAi technology. At present, in addition to gene silencing, Cloud-Clone Corp also has carried out gene overexpression and gene knockout project. Three projects go hand in hand, all have achieved remarkable results.
For more relevant products, please check www.cloud-clone.com/.