Navigation Links
Housekeeping Genes: Universal Positive,,,Controls in siRNA Knockdown Experiments

RNA interference is a powerful technique for gene knockdown experiments in academic research and an important tool for target identification and target validation in therapeutic research.

A growing number of different cell lines are used for gene knockdown experiments, and in many cases protocols for transfection do not exist. In order to optimize transfection conditions for a new cell line being investigated, luciferase-expressing plasmids are often cotransfected with the siRNA. However, the transfection conditions for plasmids and siRNA are quite different, as the plasmids must enter the nucleus, whereas siRNAs act in the cytoplasm therefore, these optimizations may lead to poor results. In addition, the use of fluorescently labeled siRNAs can give unpredictable results due to hydrophobic effects of fluorophore groups.

We have successfully used housekeeping genes as universal positive control genes for knockdown experiments in two different cell lines. The mRNA levels were analyzed using the LightCycler Instrument and were quantified relative to other housekeeping genes using the LightCycler h-Housekeeping Gene Sets.

Materials and Methods

Cell culture
Cells of the human adherent cell line PC3 were cultured in RPMI 1640 medium containing 10% (v/v) fetal calf serum and 2mM L-glutamine. Cells of the human adherent cell line HCT116 were cultured in McCoys 5A medium containing 10% (v/v) fetal calf serum and 2 mM L-glutamine.

Two days prior to transfection with siRNA oligonucleotides, 5x104 cells were plated in each well of 24- well plates. The siRNA (Dharmac on) was mixed with diluted transfection reagent as illustrated in Figures 1 and 3. The complexes were then applied to the cells in a 100 nM siRNA concentration in medium without serum (Opti-MEM), according to the manufacturers instructions. After four hours of transfection, fetal calf serum was added to 10% (v/v) final concentration.



Isolation of mRNA, synthesis of cDNA, and quantitative real-time PCR

The mRNA isolation was performed using the MagNA Pure LC Instrument. For cDNA synthesis, the 1st Strand cDNA Synthesis Kit was used, and quantitative real-time PCR was performed using the LightCycler FastStart DNA Master Hybridization Probes Kit as described in the preceding article (see pages 46).

Results

siRNA specific for the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT siRNA, sense sequence 5′CUGUCAUUAGUGAAACUGGAA, antisense sequence 5′CCAGUUUCACUAAUGACACAA) was used for a knockdown in PC3 cells. The mRNA was isolated 72 hours after transfection, transcribed into cDNA and analyzed using the LightCycler Instrument (Figure 1). Even when varying numbers of cells were used for mRNA purification (as can be seen in the differences in the crossing-point [CP] values of the housekeeping gene 5-aminolevulinate synthase) the knockdown can still be calculated by normalization to this gene.



A shift in the CP values of 2.77 was observed for the HPRT siRNA compared with the control luciferase siRNA. The knockdown efficiency was 85% (Figure 2). Cytotoxic side effects of the transfection ranging from 30% to 43% were detected using the Cell Proliferation Reagent WST-1 (compared with the control without transfection) .



siRNA specific for the housekeeping gene glucose-6- phosphate dehydrogenase (G6PDH siRNA, sense sequence 5′AACUCCUAUGUGGCUGGCCAGUU, antisense sequence 5′CUGGCCAGCCACAUAGGAGUUUU) was used for a knockdown in HCT116 cells. The mRNA was isolated 72 hours after transfection, transcribed into cDNA and analyzed using the LightCycler Instrument (Figure 3). Different transfection reagents and concentrations were applied in transfection (Figure 4). The transfection reagent R4 (currently in developement at Roche Applied Science) was the most suitable for efficient gene knockdown with low cytotoxicity.



Conclusion

The housekeeping genes G6PDH and HPRT can be used as a positive control for gene knockdown experiments when siRNA transfection conditions have to be optimized. The LightCycler Instrument allows measurement of mRNA levels relative to the mRNA levels of housekeeping genes available in the LightCycler h-Housekeeping Gene Sets.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Low Abundance cDNA Cloned Using Stratagenes Human Universal cDNA Library
2. Human Universal cDNA Library Array I
3. Universal Guide to DNA Standards
4. Custom and library siRNA for efficient gene silencing
5. Custom and library siRNA for efficient gene silencing
6. Cancer siRNA Oligo Set Version 1.0
7. Library siRNA
8. Custom siRNA Oligo Synthesis Service
9. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
10. Quantification of siRNA Silencing Efficiency Using the LightCycler System
11. Confirming gene silencing mechanism by pGFP/GFP22 siRNA co-transfection
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:12/8/2016)... OXFORD, England , December 8, 2016 ... Company, has expanded its customisable SureSeqâ„¢ NGS panel range with ... - allowing fast and cost-effective study of variants in familial ... copy number variation (CNV) detection on a single small panel ... and hotspot content. This includes all exons for LDLR ...
(Date:12/8/2016)... CA (PRWEB) , ... December 08, 2016 , ... ... as finalists in the World Technology Awards. uBiome is one of just six ... across all categories. , In addition to uBiome, companies nominated as finalists in ...
(Date:12/8/2016)... , Dec. 8, 2016  HedgePath Pharmaceuticals, ... that discovers, develops and plans to commercialize innovative ... shares of common stock were approved for trading ... begin trading on the OTCQX, effective today, under ... for the OTCQX market, companies must meet high ...
(Date:12/8/2016)...   Biocept, Inc . (NASDAQ: ... actionable liquid biopsy tests to improve the management ... its Target Selectorâ„¢ Circulating Tumor Cell platform demonstrated ... of actionable biomarkers in patients with metastatic breast ... Cannon Research Institute (SCRI), the research arm of ...
Breaking Biology Technology:
(Date:11/30/2016)... and WARSAW, Poland , Nov. 30, 2016 Not many ... of the most crucial aspects of recovery so we need to do it well. ... risks, including heart problems, high blood pressure, stroke, diabetes, and even cancer. ... and find a Christmas present that could help them to manage their sleep quality? ... ...
(Date:11/29/2016)... , Nov. 29, 2016   Neurotechnology ... and object recognition technologies, today released FingerCell ... fingerprint recognition solutions that run on low-power, ... template using less than 128KB of memory, ... devices that have limited on-board resources, such ...
(Date:11/24/2016)... 2016 Cercacor today introduced Ember TM ... non-invasively measure hemoglobin, Oxygen Content, Oxygen Saturation, ... Rate in approximately 30 seconds. Smaller than a smartphone, ... access to key data about their bodies to help ... Hemoglobin carries oxygen to muscles. When ...
Breaking Biology News(10 mins):