PCR产物直接转染细胞并转录siRNA，诱导RNAi

生物谷

编者按：RNAi是目前研究的热点，作为一种快速silence基因的重要方法。但如何制备siRNA是主要问题之一，目前至少有以下五种方法：化学合成（价格贵），体外转录制备siRNA;体外转录合成长链RNA，通过dicer或RNase III降解为短片段的siRNA；siRNA表达载体在体内生成siRNA；PCR体外扩增siRNA表达框直接转染细胞。近来越来越倾向后面几种方法，价格相对便宜，而且效果不错。下面是Ambion公司的PCR扩增siRNA表达框的一个重要产品的概述silencer expression kits,内容不错可以参考的。

Overview of Silencer™ Express Procedure

Preparing an siRNA Expression Cassette (SEC) is a simple three step process (Figure 1, below):

(I) A precursor SEC is generated by PCR using reagents in the Silencer Express Kit and one or two gene-specific oligonucleotides. The precursor SEC comprises an RNA Pol III Promoter and an adjacent hairpin siRNA template.

(II) The precursor SEC is used as a template in a large scale PCR to generate the final SEC.

(III) In the final step, the SEC is column-purified to remove contaminating oligonucleotides, dNTPs, enzymes, and salts to ensure efficient transfection. The entire procedure requires approximately one day to prepare material for transfection. Oligonucleotides encoding siRNA templates for SEC preparation can either comprise a single, long oligonucleotide or two slightly shorter oligonucleotides. The advantage of using the two-oligonucleotide approach is that the yield of SEC tends to be greater and more consistent, probably because the quality of the two shorter oligonucleotides tends to be higher.

The following steps are used in the recommended two oligonucleotide approach. (For detailed protocols for this and a single oligonucleotide approach, see the Silencer Express Instruction Manual.)

Step 1) Design one oligonucleotide with its 3' end complementary to the 3' end of the RNA Pol III promoter element provided with the kit and the 5' end complementary to the loop and sense strand of the desired siRNA (Figure 1a). The second oligonucleotide should then have a 5' end encoding the RNA Pol III terminator sequence followed by sequences complementary to the hairpin siRNA antisense strand and loop (Figure 1b).

Step 2) The first oligonucleotide is used in a PCR with the promoter element and promoter primer provided in the Silencer Express Kit.

Step 3) The resulting PCR product is then diluted and a fraction is added to a second PCR with the antisense oligonucleotide and promoter primer.

Step 4) The resulting amplification product is diluted and used to initiate a 100 µl PCR with the promoter and terminator primers provided with the kit to generate the SEC.

Step 5) The resulting amplification product is then column-purified to prepare for mammalian cell transfection.