果蝇RNAi的实验中双链短RNA的合成（dsRNA)方法

本文来自于哈佛大学医学院果蝇RNAi 筛选中心的经典实验方法，专门用于果蝇RNAi 实验方法。感谢哈佛大学医学院果蝇RNAi 筛选中心的支持！

A. Primer Designed dsRNA

B. Template Selection

C. PCR

D. In vitro RNA Transcription

E. dsRNA Purification, Quantification, & Storage

F. dsRNA From Clones

G. PCR

H. In vitro RNA Transcription

I. Purification, Quantification, & Storage

X. Additional References

We routinely produce dsRNA by in vitro transcription of a PCR generated DNA template containing the T7 promoter sequence on both ends (I. Primer Designed dsRNA). It is also possible to produce dsRNA using PCR generated DNA templates containing either the T7 & SP6 or the T7 & T3 promoters on either end (II. dsRNA Fom Clones). This method is less efficient, especially when working on a large scale.

** All work should be done in a sterile, RNase free environment, using only sterile, RNase free solutions and materials, and while wearing gloves do reduce contamination.

Templates can be generated by PCR on cDNA (including ESTs from BDGP), genomic DNA, or first strand RT-cDNA. Most of the dsRNA should correspond to exons but dsRNA with two or more exons interrupted by introns will also work well. We generally aim for ~500 bp products although RNAi with products ranging from 150-3000bp have been shown to work. The target sequence should not contain complete 21-mer homology to other genes or your dsRNA could be non-specific. One case has been seen where the template contained one mismatch in a 21-mer identical stretch to another gene, and still only the level of the intended target was reduced, while the close homologue was unaffected. Since the targeted region within a transcript may influence the success of RNAi, the safest approach is to use dsRNA corresponding to the 5' or 3' UTR. It is recommended t℃heck the various sequence sources (Publications, NCBI and BDGP, genomic and ESTs) t℃onfirm the primary sequence of a given gene.

We suggest using the Primer3 website for designing primers. Complementary sequences should be 20-24 nt, the Tm range between 59℃ - 61℃ and optimize against self-annealing by setting the Max Complementarity to 5 and Max 3' Complementarity to 1. After choosing the primer sequence, add the T7 promoter sequence (TAATACGACTCACTATAGGGAGA) to the 5' end.

Perform a standard 50 or 100µl PCR reaction using your selected primer sequences to amplify the region of interest. Use 1-2 µl of a 10µM primer stock, 100-200 ng DNA as template and DNA polymerase. We have successfully used GeneChoice Taq (PGC Scientifics #62-6086-01), TaqPlus (Stratagene #600210), and Herculase (Stratagene #NC9690330) in the following PCR reaction conditions:

1. 94℃ - 3 min.

2. 94℃ - 45 sec.

3. 57℃ - 30 sec.

4. 72℃ - 45 sec.

5. steps 2-4, 30x

6. 72℃ - 10 min.li