MysiRNA-Designer: A Workflow for Efficient siRNA Design

Mysara, Mohamed; Garibaldi, Jonathan M.; ElHefnawi, Mahmoud

doi:10.1371/journal.pone.0025642

Cited by 40 publications

(42 citation statements)

References 37 publications

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“…For the loop structure in the shRNA template, in order to avoid the formation of a termination signal, the TTCAAgAgA sequence was selected. Three target sequences were designed, and the expressionvector method was used to prepare the shRNA (22)(23)(24). This method uses a plasmid with a resistance marker as a vector for transfection of the shRNA into cells, so as to achieve sustained suppression of target gene expression (25,26).…”

Section: Discussionmentioning

confidence: 99%

Construction of a Bcl-2-shRNA expression vector and its effect on the mitochondrial apoptosis pathway in SW982 cells

Zhang

Xue

2017

Int J Mol Med

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Apoptosis is considered to serve an important role in the pathogenesis of rheumatoid arthritis. The aim of the present study was to construct Bcl-2-short hairpin (sh)RNA expression vectors and transfect them into human synovial sarcoma SW982 cells, in order to screen for an effective interference sequence and analyze the effects of this interference on the expression levels of Bcl-2 and other molecules associated with the mitochondrial apoptosis pathway. Three different shRNAs (Bcl-2-sh1, 2 and 3) were designed according to the human Bcl-2 mRNA target sequence and were transformed into competent DH5α Escherichia coli cells following the construction of an expression vector, which was then transfected into SW982 cells. SW982 cells were grouped into a control group (transfected with a negative control shRNA), and Bcl-2-sh1, Bcl-2-sh2 and Bcl-2-sh3 groups (transfected with Bcl-2-sh1, 2 and 3, respectively). The expression levels of Bcl-2 mRNA were detected using reverse transcription-quantitative PCR (RT-qPCR). Bcl-2-sh1 was identified as the most effective shRNA sequence for interference, and was used for subsequent experiments. The mRNA and protein expression levels of Bcl-2, Bax, CytC and Caspase-3 were detected in SW982 cells by RT-qPCR and western blotting at various time-points (48 and 72 h) following transfection with Bcl-2-sh1, in order to observe the effectiveness of this interference. Compared with the control group, the expression levels of Bcl-2 were decreased, while those of Bax, CytC and Caspase-3 were increased in Bcl-2-sh1-transfected cells (P<0.01). The interference effect was greater at 48 h than at 72 h. In summary, an effective shRNA sequence (Bcl-2-sh1) targeting the Bcl-2 gene was identified from three candidates, and was demonstrated to significantly interfere with the expression of Bcl-2, Bax, CytC and Caspase-3 when transfected into SW982 cells. The interference effect of Bcl-2-sh1 was more pronounced at 48 h than at 72 h post-transfection.

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Section: Discussionmentioning

confidence: 99%

Construction of a Bcl-2-shRNA expression vector and its effect on the mitochondrial apoptosis pathway in SW982 cells

Zhang

Xue

2017

Int J Mol Med

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“…The use of asymmetry is well established as being useful and important for selecting active siRNA sequences. Multistep workflow protocols for selecting effective siRNAs have been developed [41,42]. However, the selection algorithms themselves are based in part on using relative thermodynamic stability as the sole factor in determining sequence asymmetry.…”

Section: Discussionmentioning

confidence: 99%

Improved asymmetry prediction for short interfering RNAs

Malefyt

Vocelle

et al. 2014

The FEBS Journal

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In the development of RNA interference (RNAi) therapeutics, merely selecting short, interfering RNA (siRNA) sequences that are complementary to the messenger RNA (mRNA) target does not guarantee target silencing. Current algorithms for selecting siRNAs rely on many parameters, one of which is asymmetry, often predicted through calculation of the relative thermodynamic stability of the two ends of the siRNA. However, we have previously shown that highly-active siRNA sequences are likely to have particular nucleotides at each 5’-end, independent of their thermodynamic asymmetry. Here, we describe an algorithm for predicting highly active siRNA sequences based only on these two asymmetry parameters. The algorithm uses end sequence nucleotide preferences and predicted thermodynamic stabilities, each weighted based on training data from the literature, to rank the probability that an siRNA sequence will have high or low activity. The algorithm successfully predicts weakly- and highly-active sequences for enhanced green fluorescent protein (EGFP) and protein kinase R (PKR). Use of these two parameters in combination improves the prediction of siRNA activity over current approaches for predicting asymmetry. Going forward, we anticipate that this approach to siRNA asymmetry prediction will be incorporated into the next generation of siRNA selection algorithms.

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“…Several steps have been suggested for siRNA designing by Mysara et al. during their trials to design a novel siRNA therapy. ( Figure ).…”

Section: Mechanism Of Sirna and Its Designingmentioning

confidence: 99%

“…Effective siRNA designing becomes crucial to minimize costs and time constraints and maximize specificity. Several steps have been suggested for siRNA designing by Mysara et al [5] during their trials to design a novel siRNA therapy. (Figure 2).…”

Section: E C H a N I S M O F S I R N A A N D I T S D E S I G N I N Gmentioning

confidence: 99%

Therapeutic implications of small interfering RNA in cardiovascular diseases

Raghunathan

Patel

2012

Fundamemntal Clinical Pharma

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Cardiovascular diseases (CVDs) place a heavy burden on the economies of low- and middle-income countries. Comprehensive action requires combining approaches that seek to reduce the risks throughout the entire population with strategies that target individuals at high risk or with established disease. Small interfering RNA (siRNA) as a functional mediator for regulation of gene expression has been evaluated for potential therapeutic targets for the treatment of various cardiovascular diseases such as hypertension, atherosclerosis, heart failure etc. The present review attempts have been made to provide a brief outline of the current understanding of the mechanism of RNAi and the delivery system and describe the therapeutic application of siRNAs and their potential for treating CVDs which are taking a heavy toll on human life.

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MysiRNA-Designer: A Workflow for Efficient siRNA Design

Cited by 40 publications

References 37 publications

Construction of a Bcl-2-shRNA expression vector and its effect on the mitochondrial apoptosis pathway in SW982 cells

Construction of a Bcl-2-shRNA expression vector and its effect on the mitochondrial apoptosis pathway in SW982 cells

Improved asymmetry prediction for short interfering RNAs

Therapeutic implications of small interfering RNA in cardiovascular diseases

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