Technical Variations in Low-Input RNA-seq Methodologies

Bhargava, Vipul; Head, Steven R.; Ordoukhanian, Phillip; Mercola, Mark; Subramaniam, Shankar

doi:10.1038/srep03678

Cited by 85 publications

(90 citation statements)

References 37 publications

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“…Although the amounts of RNA used in the RT step were well within the manufacturers' specifications, we observed a significant concentrationdependent variability. A similar effect has also been identified in next-generation sequencing, where the use of limiting amounts of mRNA results in significant technical variation, with inefficient amplification of the majority of low to moderately expressed transcripts masking subtle biological differences (23 ). This is rather disconcerting, given the increasing interest in carrying out RNA expression analysis on single cells.…”

Section: Discussionmentioning

confidence: 91%

Variability of the Reverse Transcription Step: Practical Implications

et al. 2015

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Section: Discussionmentioning

confidence: 91%

Variability of the Reverse Transcription Step: Practical Implications

et al. 2015

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“…RNA is bound to an advanced silica gel membrane under optimal buffering conditions [21]. A simple two-step washing protocol ensures that PCR inhibitors such as proteins or divalent cations are completely removed, leaving high-quality RNA to be eluted in Milli-Q water [22].…”

Section: Sample Preparationmentioning

confidence: 99%

“…At present different method are used to produce fragmented genomic DNA, such as sonication, nebulization, enzymatic digestion, and hydrodynamic shearing. Each method has specific compensation and limitations [30]. Enzymatic digestion is simple and rapid, but it is frequently tricky to precisely control the fragment length distribution [31].…”

Section: Library Preparationmentioning

confidence: 99%

Next-generation sequencing technologies as emergent tools and their challenges in viral diagnostic

Singh¹

2018

biomedicalresearch

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Next-Generation Sequencing (NGS) has the range of high-throughput quickly adapted into the countless facet of viral diagnostic research. This method has been extensively applying in full genome sequencing, genetic diversity identification, transcriptomic routine diagnostic work patient care and management, and the new understanding of the interactions between viral and host transcriptome, to promote virus research. An exciting era of viral exploration has begun and will set us new challenges to understand the role of newly discovered viral diversity in both disease and health. In this review, discuss about the preparation of viral nucleic acid templates for sequencing, assay formats underlying next-generation sequencing systems, methods for imaging and base calling, quality control, data processing pipelines and bioinformatics approaches for sequence alignment, variant calling and suggestions for selecting suitable tools. Also discuss of the most important advances that the new sequencing technologies have brought to the fields of clinical virology and challenges behind it.

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“…The tissue is held by a toothed forceps and the lesion is detached from the base of the skin using a pair of fine iris or sharp gradle scissors. This method of biopsy produces small and often hypo-pigmented macule that may be hard to identify (Bolognia et al, 2003). …”

Section: Snip Biopsymentioning

confidence: 99%

“…There are six key biopsy techniques to perform biopsy of the skin. The selection to the type of technique employed depends on the type and size of lesion (Bolognia et al, 2003).…”

Section: Conventional Skin Sampling For Diagnostics In Dermatology 12mentioning

confidence: 99%

The skin microbiopsy

Lin¹

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Skin cancer is reported to incur the highest treatment costs of all cancers. The treatment cost of non-melanoma skin cancer (NMSC) was purported to be around AUD$264 million, and AUD$30 million for melanoma in 2001. Conventional skin biopsy techniques are performed to identify histopathological signs of malignancy. Suspicious lesions are often present on cosmetically sensitive areas and may be present in large numbers, making biopsies unfeasible. As a result, many atypical lesions are left untreated (leaving patients with early cancerous lesions) or needlessly removed (unnecessary scars). Prognostic biomarkers are useful for assessing borderline cases to facilitate early detection of skin cancer. The development of a novel microbiopsy device (Figure 1.1) from this Thesis enables quick, simple, minimally invasive and suture-free sampling of tiny skin samples sufficient for molecular assays.Laser cutting technologies have enabled rapid prototyping of microbiopsy devices made from medical-grade stainless steel. Through optimisation of the laser parameters, a twodimensional microbiopsy that assembles to form a three-dimensional cutting tip with a hallow chamber was successfully constructed. A quick and safe method to facilitate repeated collection over time in human patients is now possible with the microbiopsybased approach.Characterisation studies revealed approximately 1000 to 3000 cells from each velocityaided microbiopsy with an average DNA and RNA yield of 5.9 ± 3.4 ng and 9.0 ± 10.1 ng, respectively. Human volunteer studies have shown that the microbiopsy application ii velocity, channel width, edge surface roughness and geometries were important aspects to achieve reproducible tissue sampling.Genomic profiling of an individual can now be done using microbiopsy sampling approach instead of conventional blood or saliva samples. The limitation of small sample size can be overcome by using high-fidelity amplification technologies for amplifying nucleic acids. The defects left by microbiopsy application observed in histopathological sections were similar to artefacts arising from routine tissue processing. This opens up opportunities for the possibility of in vivo sampling and enables molecular diagnosis without the risk of damaging the lesion -which will help to improve patient care by reducing unnecessary excision. Proof-of-concept studies have established the potential of the guided skin microbiopsy device to study in vivo molecular changes within a targeted area over time, which was impossible in the past.Other than clinical applications, the microbiopsy device also offers a novel approach for live cell assays. Current approaches, in particular those related to the cosmeceutical arena are limited to restrictive regulations on animal testing, and the fact it is often not deemed ethical or practical to use conventional biopsy techniques to assess the effect of cosmetics. One debatable topic in this arena is the likelihood of nanoparticles to infiltrate the viable epidermis and cause cell toxicity. Human...

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Technical Variations in Low-Input RNA-seq Methodologies

Cited by 85 publications

References 37 publications

Variability of the Reverse Transcription Step: Practical Implications

Variability of the Reverse Transcription Step: Practical Implications

Next-generation sequencing technologies as emergent tools and their challenges in viral diagnostic

The skin microbiopsy

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