Expressions of RNAi

  • In vitro expression

    The first three methods mainly prepare siRNAs in vitro, and require specialized RNA transfection reagents to transfer siRNAs into cells. The use of siRNA expression vectors and PCR-based expression frameworks belongs to the in vivo transcription of siRNAs from DNA templates transfected into cells. The advantage of these two methods is that there is no need to manipulate RNA directly.

    siRNA expression vector

    Most siRNA expression vectors rely on one of the three RNA polymerase III promoters (pol Ⅲ) to manipulate the expression of a short hairpin RNA (shRNA) in mammalian cells. These three types of promoters include the familiar human and murine U6 promoters and human H1 promoters. The RNA pol Ⅲ promoter is used because it can express more small RNA molecules in mammalian cells, and it terminates transcription by adding a string of (3 to 6) U. To use this type of vector, you need to order 2 single strands of DNA encoding a short hairpin RNA sequence, anneal, and clone into the pol Ⅲ promoter downstream of the corresponding vector. Since cloning is involved, this process takes several weeks or even months, and it also needs to be sequenced to ensure that the cloned sequence is correct.

    The advantage of siRNA expression vectors is that they can be studied for a longer period of time-vectors with antibiotic markers can continue to inhibit the expression of target genes in cells for several weeks or even longer.

    Viral vectors can also be used for siRNA expression. Its advantage is that it can directly infect cells with high efficiency for gene silencing research, avoid all the inconveniences caused by low plasmid transfection efficiency, and the transfection effect is more stable.

    Best for: Knowing an effective siRNA sequence requires gene silencing for a long time.

    Not suitable for: screening siRNA sequences (in fact, it mainly refers to the more time-consuming and tedious work such as multiple cloning and sequencing).

    siRNA expression framework

    siRNA expression cassettes (SECs) are a kind of siRNA expression templates obtained by PCR, including an RNA pol Ⅲ promoter, a hairpin structure siRNA, and an RNA pol Ⅲ termination site, which can be directly introduced into cells for expression. No need to clone into the vector beforehand. Unlike siRNA expression vectors, SECs do not require time-consuming steps such as vector cloning and sequencing, and can be obtained directly by PCR instead of one day. Therefore, SECs have become the most effective tool for screening siRNA, and can even be used to screen the most suitable combination of promoter and siRNA in a specific research system. If restriction sites are added at both ends of the PCR, the most effective siRNA screened by SECs can be directly cloned into the vector to construct the siRNA expression vector. The constructed vector can be used for the research of stable expression of siRNA and long-term inhibition.

    The main disadvantage of this method is that ① PCR products are difficult to transfect into cells (the protocol of Jingsai Company can solve this problem) ② Sequence determination cannot be performed, and misreading that may be poor during PCR and DNA synthesis cannot be found, leading to unsatisfactory results. .

    Best for: Screening siRNA sequences, screening the best promoter before cloning into the vector

    Not applicable: long-term inhibition studies. (It will be fine if it is cloned into the vector)

    RNA interference main experiment

    After the siRNA expression vector is constructed, the main RNA interference experiment can be carried out.

     

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