Figure 1. Silencing Gene Targets by RNase III Derived siRNA Cocktails. A
200 bp dsRNA (15 μg) for each gene of interest was digested with 2.5 U
RNase III for 1 hour at 37℃. 1A. RNase III efficiently digests dsRNA .
One microgram of the dsRNA before and after RNase III digestion was run
on a 15% non-denaturing acrylamide gel along with a 21 bp chemically
synthesized siRNA to GAPDH, which served as a size marker. The gel was
stained with ethidium bromide and photographed under UV light. 1B. RNase
III derived siRNA cocktails silence GAPDH, La and c-FOS. GAPDH and La
siRNA cocktails were transfected into HeLa cells. The c-fos siRNA
mixture was transfected into 293 cells followed by 24 hours of
stimulation with 50 nM PMA. All samples were harvested at 48 hours post
transfection and immunofluorescence was performed with the appropriate
antibodies. Fluorescence signal was quantitated, normalized for cell
number and graphed.
12-15bp 的RNase III 降解產物誘導基因沉默的效果
最常用于介導RNA i 的化學合成siRNA 通常是21bp 大小。已經有實驗結果證實：用RNase III 在特定條件下降解得到的21bp
大小產物是非常有效的基因表達抑制劑。然而RNase III 完全消化產物是12―15bp 大小的片斷，為了要研究這些12-15bp
大小的片斷抑制基因表達的效果，一個200bp 的GAPDH 雙鏈RNA 用RNase III 完全消化得到12-15bp
的產物并用聚丙烯酰胺膠純化，按100nM 濃度轉染Hela 細胞，另外化學合成的已知能有效抑制GAPDH 表達的siRNA
序列在同樣濃度下轉染Hela 細胞作為對照，相應的免疫熒光圖象表明：12-15bp
的降解產物也能夠抑制基因的表達，在相同濃度下兩種方法的抑制效果相當。這個結果表明由RNase III 制備的更短一些的siRNA
混合庫能夠經過轉染哺乳動物細胞抑制靶基因的表達，似乎沒有必要通過改變反應條件或者借助純化手段制備較長的siRNA
從而抑制靶基因的表達。改變轉染的濃度，結果顯示siRNA 的轉染劑量對基因抑制效果有直接影響，RNase III 制備的siRNA
庫需要保持較高的濃度從而達到最大的抑制效果，原因可能是由于：在這個siRNA s 庫中既有一些有效的siRNA s
分子，也有很多無效的siRNA s 部分。
Figure 2. 12-15 bp RNase III Digestion Products Elicit Silencing. A 200
bp GAPDH dsRNA (30 μg) was digested with RNase III(30 U) for 1 hour at
RT. Digestion products were run on a 15% non-denaturing acrylamide gel
and the 12-15 bp products were excised, eluted, and ethanol
precipitated. A sample was run on a 15% non-denaturing acrylamide gel
for visualization (2A). HeLa cells were transfected with 100 nM of the
12-15 bp RNase III generated GAPDH siRNA s or a 21 bp chemically
synthesized GAPDH siRNA . GAPDH protein levels were monitored by
immunofluorescence 48 hours after transfection (2B) and the resulting
images were quantitated (2C).
基因沉默的專一性
進一步的實驗來驗證這種方法制備的siRNA 庫抑制靶基因表達的專一性：用RNase III 制備的針對GAPDH 的siRNA 庫轉染Hela
細胞，在降低GAPDH 表達的同時檢測其他一系列非特異基因 (La, Ku-70, c-myc, ?-actin, and cdk-2)
的表達水平，結果沒有檢測到這些非特異基因的表達在轉染前后的變化。結果顯示在轉染RNase III 制備的siRNA s
庫后沒有發生非特異基因沉默。 最近一篇關于RNase III 制備的siRNA s 和相關的RNA
結合蛋白的文章同樣證實沒有非特異基因沉默的發生 (5) 。在除了哺乳動物細胞以外的其他系統中，可以通過Dicer 酶復合物消化長片斷RNA
雙鏈，得到針對同一個靶基因多個位點的siRNA s 群，從而特異的抑制目的基因的表達。這些結果提示存在某種適當的機制來維護抑制反應的高度專一性。
結論
用RNase III 消化dsRNA 制備siRNA
庫從而有效抑制目的基因表達，這是一種比較簡單粗放、快速、性價比很高的方法。根據實驗結果，導入這種方法制備的siRNA
庫，并沒有表現比用化學合成法制備特定的siRNA s 更高的毒性或者是對基因表達的非特異效應。采用RNase III 制備siRNA s
混合庫可以避免化學合成siRNA 的昂貴費用，siRNA 表達載體的繁瑣勞動，以及為找到一個有效的siRNA
序列所必經的漫長的篩選過程，因而成為有別于傳統方法的制備siRNA 的另一種好辦法，特別是對于只需要運用RNA i
技術作為遺傳工具，快速得到RNA 干擾結果，而無需詳細研究siRNA 具體信息的研究人員更是如此。
Figure 3. RNase III siRNA Cocktails Show Specificity for Silencing.
HeLa cells were transfected with 100 nM RNase III generated siRNA s to
GAPDH. Immunofluorescence analysis of GAPDH, La, c-MYC, Cdk-2, Ku-90,
and ?-actin was performed 48 hours post transfection and subsequently
quantitated.
References
1. Byrom MW, Cheng AM, Ford LP. (2003) Characterizing RNA i induced
with siRNA cocktails generated by RNase III. Ambion TechNotes 10(1):
4-6.
2. Yang D, Buchholz F, Huang Z, Goga A, Chen CY, Brodsky FM, Bishop JM.
(2002) Short RNA duplexes produced by hydrolysis with Escherichia coli
RNase III mediate effective RNA interference in mammalian cells. Proc
Natl Acad Sci USA 99(15): 9942-7.
3. Calegari F, Haubensak W, Yang D, Huttner WB, and Bushholz F. (2002)
Tissue-specific RNA interference in postimplantation mouse embryos with
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C, santilli G, Byrom MW, Goldoni S, Ford LP, Caligiuri MA, Maraia RJ,
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