關(guān)鍵詞： RNAi

編者按：RNAi是目前研究的熱點(diǎn),，作為一種快速silence基因的重要方法。但如何制備siRNA是主要問題之一,，目前至少有以下五種方法：化學(xué)合成（價(jià)格貴）,，體外轉(zhuǎn)錄制備siRNA;體外轉(zhuǎn)錄合成長(zhǎng)鏈RNA，通過dicer或RNase III降解為短片段的siRNA,；siRNA表達(dá)載體在體內(nèi)生成siRNA,；PCR體外擴(kuò)增siRNA表達(dá)框直接轉(zhuǎn)染細(xì)胞。近來(lái)越來(lái)越傾向后面幾種方法，價(jià)格相對(duì)便宜,，而且效果不錯(cuò),。下面是Ambion公司的PCR擴(kuò)增siRNA表達(dá)框的一個(gè)重要產(chǎn)品的概述silencer expression kits,內(nèi)容不錯(cuò)可以參考的。

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.

 
Figure 1. Schematic of Preparing SECs using Two siRNA Template Oligonucleotides.