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The following Cold Spring Harbor Laboratory Press protocols are brought to you by BioSupplyNet in a partnership with Sigma-Aldrich. A list of products is available to use in each protocol.
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View the protocol "Production of Endoribonuclease-Prepared Short Interfering RNAs (esiRNAs) for Specific and Effective Gene Silencing in Mammalian Cells" at BioSupplynet.com
Anne-Kristin Heninger and Frank Buchholz
ABSTRACT
The mechanism of RNA interference has emerged as a practical tool to study loss of function in many organisms. To make the method of gene knockdown suitable for mammalian cells, short interfering double-stranded RNAs (siRNAs) need to be applied. This protocol describes a straightforward, low-cost method, which uses recombinant Escherichia coli RNase III to digest long dsRNA into endoribonuclease-prepared short interfering RNAs (esiRNAs). Advantages of this technology are the high efficiency and specificity of the resulting esiRNA and its usefulness for not only small-scale applications, but also high-throughput loss-of-function analyses. Another great asset of esiRNA is its flexibility in design, using Web-based tools such as DEQOR, or predesigned esiRNA sequences from the database RiDDLE. PCR products flanked with T7 promoter sequences are generated, transcribed, and annealed. The resulting long dsRNA is enzymatically digested into a pool of overlapping esiRNAs, which are subsequently spin-column-purified.
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| Products Available for this Protocol |
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Agarose |
A4718 |
Agarose, High resolution, DNase, RNase, NICKase, none detected |
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| dNTP mix |
D7295 |
Deoxynucleotide Mix, 10 mM, Molecular Biology Reagent |
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| Tris-Cl |
T5941 |
Trizma® hydrochloride, for molecular biology, Biotechnology Performance Certified, cell culture tested, ≥99.0% (titration) |
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| EDTA |
E5134 |
Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecular biology, ≥99% |
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| MgCl2 |
M4880 |
Magnesium chloride, insect cell culture tested |
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| DTT |
D9779 |
DL-Dithiothreitol, for molecular biology, ≥98% (TLC), ≥99% (titration) |
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| NaCl |
S6191 |
Sodium chloride, Biotechnology Performance Certified, ≥99.5% (titration), Cell Culture Tested |
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| KCl |
P9541 |
Potassium chloride, for molecular biology, ≥99% |
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| Glycerol |
G5516 |
Glycerol, for molecular biology, ≥99% |
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| H2O |
W4502 |
Water, Molecular Biology Reagent |
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| Ethanol |
E7148 |
Ethanol, for molecular biology, 190 proof |
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| Isopropanol |
I9782 |
Isopropyl alcohol, meets EP, BP, USP testing specifications |
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| Ficoll |
F2637 |
Ficoll®, for molecular biology, lyophilized powder |
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| Orange G (acid orange 10) |
O7252 |
Orange G, certified by the Biological Stain Commission |
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| DNA marker with fine resolution (e.g., 25-bp DNA ladder) |
P1598 |
PCR 20 bp Low Ladder, buffered aqueous solution |
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| Red DNA polymerase |
D2812 |
REDTaq® Genomic DNA Polymerase, without MgCl2 |
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| Q-Sepharose FastFlow |
Q1126 |
Q Sepharose® Fast Flow, preswollen, 45-165 µm (wet), pore size ~4,000,000 Da exclusion limit |
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Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.
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