Skin Tissue Surface Morphology and Quality of RNA and Protein Extracted from Fresh and Stabilized Human Cleft Lip and Palate Tissue

Shah, Nurul Syazana Mohamad; Sulaiman, Wan Azman Wan; Halim, Ahmad Sukari; Sulong, Sarina

doi:10.3889/mjms.1857-5773.2014.0351

Cited by 2 publications

(2 citation statements)

References 26 publications

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“…In previous studies, the correlation between tissue weight and RNA yield has been controversial. In the case of human (upper thigh or arm) and mouse (back) skin tissues, a linear relationship has been demonstrated 20 , while another study found no such correlation when analysing human cleft lip skin tissue 21 . The lack of correlation between tissue input and RNA output and the difference between individuals in the case of pigs showed that other factors might affect the RNA yield in addition to tissue weight.…”

Section: Discussionmentioning

confidence: 95%

Identification of an optimal method for extracting RNA from human skin biopsy, using domestic pig as a model system

Reimann

Abram

Kõks

et al. 2019

Sci Rep

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To evaluate skin tissue gene expression patterns correctly, extracting sufficient quantities of good quality RNA is essential. However, RNA extraction from skin tissue is challenging, as the hyaluronic acid-collagen matrix is extremely difficult to homogenize. Although there are multiple ways to extract RNA from skin, there are no comparative studies that identify the most critical steps, e.g. sample collection, storage and homogenization. We analysed the various steps involved in RNA extraction (i.e. biopsy collection as dry biopsy or into nucleotide stabilizing reagents, different storage conditions, enzymatic digestion, stator-rotor and bead motion-based homogenizing combined with column-based RNA purification). We hypothesised that domestic pig skin is applicable as a model for human skin studies. Altogether twenty different workflows were tested on pig skin and the four most promising workflows were tested on human skin samples. The optimal strategy for extracting human skin RNA was to collect, store and homogenize the sample in RLT lysis buffer from the RNeasy Fibrous Tissue Kit combined with beta-mercaptoethanol. Both stator-rotor and bead motion-based homogenizing were found to result in high quality and quantity of extracted RNA. Our results confirmed that domestic pig skin can be successfully used as a model for human skin RNA studies.

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Section: Discussionmentioning

confidence: 95%

Identification of an optimal method for extracting RNA from human skin biopsy, using domestic pig as a model system

Reimann

Abram

Kõks

et al. 2019

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Portillo et al (2006) reported that differences in the RNA concentration and quality, among samples or treatments, may result from differences in the extraction efficiency and/or quality of the RNA. Furthermore, Syazana et al (2014) reported that if RNA yield is lower than expected, the homogenization may not have been successfully completed or maceration of the material may not have been properly performed. These steps often vary, depending on the type of sample used for extraction and the type and initial volume of the lysis buffer utilized.…”

Section: Discussionmentioning

confidence: 99%

DNase concentration assay to obtain DNA-free RNA from sugarcane leaves

et al. 2016

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The success of gene expression studies, protein synthesis, and construction of cDNA libraries directly depends on the purity and integrity of the RNA used in these tests, as even minor amounts of contaminant DNA (<1%) can produce a false positive amplification signal in quantitative real-time PCR. For RNA contaminated with genomic DNA, an essential step in the studies on gene expression is the treatment of the RNA samples with DNase. This study was conducted to test three different concentrations of DNase I (0.02, 0.04, and 0.06 μL/ng of RNA), which were chosen based on the results of the RNA sample quantifications and as indicated by the manufacturer, to digest genomic DNA present in the RNA samples extracted from sugarcane leaves with the Concert™ Plant RNA Reagent. The results showed that all three concentrations of DNase significantly reduced DNA concentrations. However, RNA was also degraded on DNase I treatment. In addition, the amount of DNA present in the RNA samples after purification with DNase I was sufficient for its amplification in the tests conducted with conventional PCR. Furthermore, the condition of RNA samples obtained after the treatments allowed for real-time PCR. Therefore, we concluded that 0.02 μL DNase I was the ideal concentration for sugarcane RNA purification, as higher concentrations do not increase the efficiency of the genomic DNA digestion in RNA samples and only make the purification process more expensive. This study provides important information on the effect of high concentrations of DNase I and complements previous studies that have so far tested only the DNase concentration recommended by the manufacturer.

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Skin Tissue Surface Morphology and Quality of RNA and Protein Extracted from Fresh and Stabilized Human Cleft Lip and Palate Tissue

Cited by 2 publications

References 26 publications

Identification of an optimal method for extracting RNA from human skin biopsy, using domestic pig as a model system

Identification of an optimal method for extracting RNA from human skin biopsy, using domestic pig as a model system

DNase concentration assay to obtain DNA-free RNA from sugarcane leaves

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