Navigation Links
Selecting siRNA Sequences to Incorporate into the pSilencer Vectors


The first step in preparing a plasmid for siRNA experiments is to identify target sequences in the gene of interest that are susceptible to siRNA-induced degradation. We have found that a little more than half of the siRNAs provide at least a 50% reduction in target mRNA levels and approximately 1 out of 4 siRNAs provide a 75-95% reduction. The general process begins with scanning the length of the target gene for potential siRNA target sites. For siRNA expression vectors, the target sites should have 5' terminal AAs because, upon folding the siRNAs hairpins will give rise to UU overhangs. These UU overhangs will therefore be complementary to the AA in the target site. The 21 nucleotide siRNA target sequences are then compared to an appropriate genome database to eliminate sequences with significant homology to other genes. For screening, we typically test four siRNAs per target. We space the siRNAs down the length of the gene sequence to reduce the chances of targeting a region of the mRNA that is either highly structured or bound by regulatory proteins.

Constructing four siRNA expression plasmids for each target can be time-consuming and expensive. In vitro transcription provides a less formidable siRNA screening method. To ensure that siRNAs expressed from plasmids are functionally equivalent to siRNAs prepared by in vitro transcription, we prepared plasmids and siRNAs targeting four different sequences in the cyclophilin and GAPDH genes. These nucleic acids were transfected into HeLa cells. Silencing was evaluated by Northern analysis using probes specific to GAPDH, cyclophilin, and 28S rRNA. The hybridiz ation signal from the various targets was quantitated by phosphorimager. As seen in Figure 1, the susceptibility of siRNA target sites to siRNA-mediated gene silencing appears to be comparable for both in vitro prepared siRNAs and RNA Pol III-expressed siRNAs. This is also true for chemically synthesized siRNAs versus RNA Pol III-expressed siRNAs. Therefore, it is not necessary to re-screen genes for which functional siRNAs have already been identified.

Transcribed siRNA siRNAVector GAPDH None 100% 100% 5' 25% 17% 5' Medial 7% 13% 3' Medial 36% 20% 3' 45% 63% Cyclophilin None 100% 100% 5' 5% 23% 5' Medial 15% 19% 3' Medial 45% 29% 3' 70% 54%

Figure 1. Correlation of Target Site Selection Between siRNA Generated In Vitro and In Vivo. siRNAs to four target sites in each of two genes (GAPDH and cyclophilin) were selected and prepared using the Silencer siRNA Construction Kit. Sequences encoding hairpin siRNAs to the same target sequences were sub-cloned into pSilencer 2.0-U6 and 3.0-H1. The siRNAs and siRNA expression vectors were transfected into HeLa cells using siPORT-Lipid (Ambion). Target RNA levels were assessed post-transfection (48 hr) by Northern analysis using the NorthernMax procedure (Ambion). The relative levels of the target genes were measured against cells transfected with negative control siRNA or negative control siRNA expression vector.


back to top


Ordering Information
Cat# Product Name Size 1620 Silencer siRNA Construction Kit 15 siRNA synthesis rxns 7207 pSilencer 1.0-U6 (circular) 20 g 7208 pSilencer 1.0-U6 (linear) 20 rxns 7209 pSilencer 2.0-U6 20 rxns 7210 pSilencer 3.0-H1 20 rxns
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Custom and library siRNA for efficient gene silencing
2. Custom and library siRNA for efficient gene silencing
3. Cancer siRNA Oligo Set Version 1.0
4. Library siRNA
5. Custom siRNA Oligo Synthesis Service
6. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
7. Quantification of siRNA Silencing Efficiency Using the LightCycler System
8. Housekeeping Genes: Universal Positive Controls in siRNA Knockdown Experiments
9. Confirming gene silencing mechanism by pGFP/GFP22 siRNA co-transfection
10. siRNA transfection optimization with the Agilent 2100 bioanalyzer
11. siRNA Design Guidelines
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/24/2016)... ... June 24, 2016 , ... Researchers at the Universita ... miRNAs in people with peritoneal or pleural mesothelioma. Their findings are the subject of ... now. , Diagnostic biomarkers are signposts in the blood, lung fluid or tissue ...
(Date:6/23/2016)... 23, 2016   Boston Biomedical , an ... designed to target cancer stemness pathways, announced that ... Orphan Drug Designation from the U.S. Food and ... cancer, including gastroesophageal junction (GEJ) cancer. Napabucasin is ... inhibit cancer stemness pathways by targeting STAT3, and ...
(Date:6/23/2016)... 23, 2016 Houston Methodist Willowbrook Hospital ... Sports Association to serve as their official health ... Methodist Willowbrook will provide sponsorship support, athletic training ... association coaches, volunteers, athletes and families. ... Sports Association and to bring Houston Methodist quality ...
(Date:6/23/2016)... -- The Biodesign Challenge (BDC), a university competition that asks ... systems and biotechnology, announced its winning teams at the ... York City . The teams, chosen ... MoMA,s Celeste Bartos Theater during the daylong summit. Keynote ... of architecture and design, and Suzanne Lee , ...
Breaking Biology Technology:
(Date:4/15/2016)... , April 15, 2016  A new partnership ... more accurate underwriting decisions in a fraction of ... competitively priced and high-value life insurance policies to ... With Force Diagnostics, rapid testing (A1C, ... data readings (blood pressure, weight, pulse, BMI, and ...
(Date:4/13/2016)... CHICAGO , April 13, 2016  IMPOWER physicians ... are setting a new clinical standard in telehealth ... By leveraging the higi platform, IMPOWER patients can ... weight, pulse and body mass index, and, when they ... quick and convenient visit to a local retail location ...
(Date:3/29/2016)... , March 29, 2016 ... "Company") LegacyXChange "LEGX" and SelectaDNA/CSI Protect are pleased to ... ink used in a variety of writing instruments, ensuring ... of originally created collectibles from athletes on LegacyXChange will ... analysis of the DNA. Bill Bollander ...
Breaking Biology News(10 mins):