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An optimized microRNA scaffold increases shRNA processing and target knockdown

ABSTRACT: An optimized microRNA scaffold increases shRNA processing and target knockdown

PROVIDER: PRJNA263394 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR1608481.fastq.gz	Fastqsanger.gz
	SRR1608482.fastq.gz	Fastqsanger.gz
	SRR1608483.fastq.gz	Fastqsanger.gz
	SRR1608484.fastq.gz	Fastqsanger.gz
	SRR1608485.fastq.gz	Fastqsanger.gz

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An optimized microRNA scaffold increases shRNA processing and target knockdown

Project description:Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels). Purpose: Others have reported that the EcoRI site that was introduced to the mir30 scaffold results in decreased smallRNA processing and hence reduced target knockdown. We've developed an alternative scaffold (termed ultramir) where this site is removed. smallRNA cloning was used to determine if the movement of this sight resulted in an increase in shRNA processing. Method: Two shRNAs (one targeting Renilla Luciferase and one targeting Human RPA3) were cloned into the original mir30 cassette the ultramir cassette. Each of the 4 constructs were infected in duplicate at single copy into cells and the cells seltected unitil infection percentages reached >90% (the shRenilla hairpin was infected into HEK293T cells and the shRPA3 construts into the Gallus gallus cell line ERC. After selection smallRNA cloning was perfromed and the amount of smallRNAs corrresponding to the two shRNAs compared to the endogenous microRNA populatlon. Results: smallRNA levels of the two shRNAs doubled relative to the microRNA population when they were placed into the ultramir scaffold.

2014-11-24 | E-GEOD-62187 | biostudies-arrayexpress

An optimized microRNA scaffold increases shRNA processing and target knockdown

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Optimized low-volume Plasmodium blood sample processing protocols for untargeted transcriptomics

Project description:As the importance of transcriptional variation and regulation for Plasmodium becomes more apparent, advances for non-falciparum species are hindered by our reliance on natural infections to study parasite biology. Untargeted transcriptomic research is also complicated by low parasite densities and high proportions of human genetic material, highlighting the need for optimized sample processing protocols. In this study, we used a P. knowlesi culture diluted in whole blood as a mock P. vivax natural infection to compare white blood cell, rRNA-, and globin depletion methods and RNA-seq library preparation kits to create an optimized protocol for low-volume sample processing.

2025-04-16 | GSE294674 | GEO

Sub-genomewide shRNAs constructed using an optimized selection algorithm and microRNA backbone provide stronger evidence for follow-up studies

Project description:Sub-genomewide shRNA libraries were constructed using the current RNAi consortium constructs as well as using the DSIR (siRNA algoirthm) and a novel shRNA specific algorithm (shERWOOD). All libraries were placed into mir30 expression vectors. The shERWOOD libraries were also placed in a vector harboring an optimized mir cassette (ultramir). Each library was screened using the pancreatic cell line A385. A concensus set of essential genes identified as the set for which two shRNAs depleted in each of the libries. For these genes, a great percentage of shERWOOD seletected shRNA depleted. In addition the placement of shERWOOD selected constructs into ultramir scaffoled increased the rate of shRNA depletion for essential genes further. Purpose: shRNA screens were carried out using various library construction strategies to identify the strategy that provides the best shRNA screening results. Method: Libraries were constructed using the TRC shRNA set as well as shRNAs identified using the DSIR and shERWOOD algorithms. shRNA libraries were cloned into mir30 expression vectors. shERWOOD shRNAs were also cloned into an expression vector harboring an optimized microRNA scaffold termed ultramir. Each resultant library was screened using the pancreatic cell line A385. Each library was analyzed separately to identify a set of genes where at least two shRNAs depleted. These gene sets were intersected to develop a set of essential genes. Results: The shERWOOD shRNA libraries provided the highest number depleting shRNAs for each essential gene. Further these shRNAs depleted to a greater extent than did the shRNAs from the other libraries. When shERWOOD libraries were placed into the ultramir cassette a greater number of shRNAs per essential gene depleted.

2014-11-24 | E-GEOD-62185 | biostudies-arrayexpress

Optimized low-volume Plasmodium blood sample processing protocols for untargeted transcriptomics

Project description:Optimized low-volume Plasmodium blood sample processing protocols for untargeted transcriptomics

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Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

Project description:Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

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Optimized periphery-core interface increases fitness of the Bacillus subtilis glmS ribozyme

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The helicase domain of Dicer ensures strict processing of evolutionarily optimized microRNAs

Project description:The helicase domain of Dicer ensures strict processing of evolutionarily optimized microRNAs