RNA Extraction Protocols Research Articles

An improved method for the rapid isolation of RNA from Arabidopsis and seeds of other species high in polyphenols and polysaccharides

AbstractSeeds are notoriously high in polyphenols and polysaccharides, which reduce RNA quality and yield, and interfere with downstream applications. We present simple modifications to a rapid RNA extraction protocol for use with seeds. The inclusion of polyethylene glycol in place of polyvinylpyrrolidone reduced polyphenol and polysaccharide contamination. In addition, replacing NaCl with KCl improved the RNA yield from Arabidopsis seeds still bound by mucilage. On extraction of Arabidopsis seed recovered from field soils clean RNA pellets with no accompanying gelatinous matrix (polysaccharide) were seen, with A260/230 ratios greater than 1.8 confirming the lack of polysaccharide carry-over. WhenBrassica oleraceaandSinapis arvensisseeds were extracted, 260/230 ratios greater than 1.8 were seen. RNA yields in excess of 10 µg per 100 mg seed suitable for RT-QPCR were obtained.

Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling.

Background: The measurement of circulating miRNAs has proven to be a powerful bi-omarker tool for several disease processes. Current protocols for the detection of miRNAs usually in-volve an RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain suffi-cient input material.Objective: Here, we describe a novel methodology that allows detection of a large number of miRNAs from a small volume of serum or plasma without the need for RNA extraction.Methods: Three μl of serum or plasma was subjected to three cycles of high and low temperatures (heat/freeze cycles) followed by miRNA arrays.Results: Our results indicate that miRNA detection following this process is highly reproducible when comparing multiple samples from the same subject. Moreover, this protocol increases the reproducibility of miRNA detection in samples that were previously subjected to multiple freeze-thaw cycles. Im-portantly, the detection of miRNAs from serum vs. plasma following heat/freeze cycling are highly comparable, indicating that this heat/freeze process effectively eliminates differences in detection be-tween serum and plasma samples that have been reported using other sample preparation methodologies.Conclusion: We propose that this method is a potent alternative to current RNA extraction protocols, substantially reducing the amount of sample necessary for miRNA detection while simultaneously im-proving miRNA detection and reproducibility

Comparison of RNA extraction and microRNA detection protocols for a small amount of germinal vesicle oocytes in bovine

RT-qPCR is a widely used method to detect miRNA expression. Compared with mRNA, miRNA has a shorter length and lower abundance which hinders the acquisition of reliable results. Thus, miRNA detection requires a method with high sensitivity and accuracy. Collecting large amounts of material is particularly difficult for oocytes and pre-implantation embryos of domestic animals. Establishing a set of miRNA detection methods that are suitable to detect trace amounts of such materials is urgently needed. In this study, the total RNA in 50 germinal vesicle (GV) oocytes was isolated through direct lysis and by using mirVana miRNA Isolation Kit and miRNeasy Micro Kit. The OD260/280 values and concentrations of the RNA in these three groups were compared to identify a superior RNA isolation method. In addition, the specificity and sensitivity of common DNA and LNA primers were compared by real-time quantitative polymerase chain reaction for miRNA detection. Results show that the RNA concentration of in the direct lysis group was significantly higher than that in the other two groups. The specificity between the DNA primers and LNA primers was identical, whereas the sensitivity of LNA primers was superior to that of DNA primers. These results suggest that direct lysis combined with LNA primers might be a suitable protocol for the miRNA detection of a small amounts of GV oocytes and pre-implantation embryos in cattle.

A novel combination of silane-coated silica colloid with hybrid RNA extraction protocol and RNA enrichment for downstream applications of spermatozoal RNA.

Spermatozoa are specialised cells with low RNA content as compared to somatic cells. The suitable sperm RNA extraction and enrichment protocols for downstream applications are available for human, cattle, stallion and mouse but not for buffalo spermatozoa. Therefore, the present work was conducted to find out suitable colloidal solution for sperm purification and appropriate protocol for sperm RNA extraction and enrichment/amplification of RNA. For purification, we used PVP-coated silica colloidal solution (PVP-Si), silane-coated silica colloidal solution (Silane-Si) and iodixanol. Sperm recovery rate, total sperm motility and progressive sperm motility were significantly improved after separation by Silane-Si and iodixanol compared to PVA-Si method. The combined guanidinium thiocyanate-phenol-chloroform (GTPC) with silica matrix (SM)-based RNA extraction yielded more quantity of RNA in compared to individual method. The hybrid of SM and GTPC into a single protocol yielded 360-450ng RNA from 30 million buffalo spermatozoa. For the first time, we adopted new way to enrich sperm RNA that increased the RNA concentration 4-5 times that was sufficient for downstream applications. The linear amplification of sperm RNA increased RNA concentration around 27-45 times. In summary, Silane-Si colloid for sperm separation, hybrid SM and GTPC protocol for sperm RNA extraction followed by enrichment or amplification of RNA was found suitable for high-throughput analyses of buffalo sperm RNA.

Selective recovery of RNAs from bacterial pathogens after their internalization by human host cells.

Selective RNA extractions are required when studying bacterial gene expression within complex mixtures of pathogens and human cells, during adhesion, internalization and survival within the host. New technologies should be developed and implemented to enrich the amount of bacterial RNAs since the majority of RNAs are from the eukaryotic host cells, requiring high read depth coverage to capture the bacterial transcriptomes in dual-RNAseq studies. This will improve our understanding about bacterial adaptation to the host cell defenses, and about how they will adapt to an intracellular life. Here we present an RNA extraction protocol to selectively enrich the lowest bacterial RNA fraction from a mixture of human and bacterial cells, using zirconium beads, with minimal RNA degradation. Zirconium beads have higher capacity to extract bacterial RNAs than glass beads after pathogen internalization. We optimized the beads size and composition for an optimal bacterial lysis and RNA extraction. The protocol was validated on two human cell lines, differentiated macrophages and osteoblasts, with either Gram-positive (Staphylococcus aureus) or -negative (Salmonella typhimurium) bacteria. Relative to other published protocols, yield of total RNA recovery was significantly improved, while host cell infection was performed with a lower bacterial inoculum. Within the host, bacterial RNA recovery yields were about six-fold lower than an RNA extraction from pure bacteria, but the quality of the RNA recovered was essentially similar. Bacterial RNA recovery was more efficient for S. aureus than for S. typhimurium, probably due to their higher protection by the Gram-positive cell walls during the early step of eukaryotic cell lysis. These purified bacterial RNAs allow subsequent genes expression studies in the course of host cell-bacteria interactions.

Transcriptomic profiling of human skin biopsies in the clinical trial setting: A protocol for high quality RNA extraction from skin tumours.

Transcriptomic profiling of skin disease using next generation sequencing allows for detailed information on aspects of RNA biology including gene expression, non-coding regulatory elements and gene splicing. The application of RNA sequencing to human skin disease and cancer is often hampered by degraded RNA. Here we describe a protocol that allows for consistently intact RNA to be extracted from snap frozen skin biopsy samples, which has been validated in a clinical trial setting. Human skin tumour punch biopsies (n=28) ranging from 4-6mm in diameter were obtained from 14 patients with an inherited skin tumour syndrome (CYLD cutaneous syndrome) and frozen in liquid nitrogen prior to being stored at -80°C. These samples were then subject to cyrostat sectioning, allowing for histological assessment, and were homogenised using a bead-based lysis platform. RNA extraction was performed using a silica column-based system. RNA concentration was measured using fluorescent quantitation and RNA integrity assessed using microfluidic gel electrophoresis. We also processed normal skin biopsies using the same protocol (n=10). The mean RNA integrity score of the tumour and normal samples was 9.5, and the quantity of RNA obtained from the small amounts of tissue used exceeded requirements for RNA-seq library generation. We propose that the method of RNA extraction suggested here allows for transcriptomic profiling from small pieces of human tissue without the need for PCR amplification during library preparation. This protocol could be utilised in healthy and diseased skin to improve mechanistic understanding in a range of human skin diseases.

MirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets

ABSTRACTTechniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method “mirRICH.” The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.

A rapid and efficient SDS-based RNA isolation protocol from different tissues of coffee.

Isolation of high-quality RNA from coffee is challenging because of high level of polysaccharides, polyphenols and other secondary metabolites. In the present study, a rapid and efficient RNA extraction protocol from different tissues of coffee was optimized. Sufficiently high quality and quantity (225.6-454.8µg/g) of RNA was obtained by using the optimized protocol. The presence of two distinct bands of 28S rRNA and 18S rRNA in agarose gel proved the intactness of the RNA samples. The average spectrophotometric values of the isolated RNA ranged from 1.96 to 2.02 (A260/280) and 1.95 to 2.14 (A260/230), indicating the high quality of RNA devoid of polyphenols, polysaccharides and protein contamination. In the optimized protocol, addition of PVPP to the extraction buffer and a brief incubation of samples at 65°C and subsequent purification with potassium acetate resulted in good-quality RNA isolation. The suitability of RNA for downstream processing was confirmed by PCR amplification with cytochrome c oxidase gene-specific primers. The amplification of a single 392bp fragment using cDNA and 1.5kb fragment using genomic DNA samples confirmed the absence of DNA contamination. The present protocol is rapid and yielded good quality and quantity of RNA suitable for functional genomics studies.

Combining genotypic and phenotypic analyses on single mutant zebrafish larvae

Zebrafish is a powerful animal model used to study vertebrate embryogenesis, organ development and diseases (Gut et al., 2017) [1]. The usefulness of the model was established as a result of various large forward genetic screens identifying mutants in almost every organ or cell type (Driever et al., 1996; Haffter et al., 1996) [2,3]. More recently, the advent of genome editing methodologies, including TALENs (Sander et al., 2011) [4] and the CRISPR/Cas9 technology (Hwang et al., 2013) [5], led to an increase in the production of zebrafish mutants. A number of these mutations are homozygous lethal at the embryonic or larval stages preventing the generation of homozygous mutant zebrafish lines. Here, we present a method allowing both genotyping and phenotype analyses of mutant zebrafish larvae from heterozygous zebrafish incrosses. The procedure is based on the genotyping of the larval tail after transection, whereas phenotypic studies are performed on the anterior part of the zebrafish larvae.•The method includes (i) a protocol for genotyping, (ii) protocols for paraffin embedding and histological analyses, (iii) protocols for protein and histone extraction and characterization by Western blot, (iv) protocols for RNA extraction and characterization by RT-PCR, and (v) protocols to study caudal spinal cord regeneration.•The technique is optimized in order to be applied on single zebrafish embryos and larvae.

Comparative analysis and innovation of a simple and rapid method for high-quality RNA and DNA extraction of kiwifruit

RNA and DNA extraction is a requirement for the study of gene expression and has an increasingly important role in genetic studies of all fleshy fruits. RNA and DNA extraction is difficult in kiwifruit due to the significant amount of polysaccharides and polyphenols compounds. So far, no commercial kit has been developed specifically for high-quality RNA and DNA extraction in kiwifruit and the common protocols for RNA extraction have poor yields. This study developed a new protocol for high quality RNA extraction in Actinidia deliciosa. According to the results, the average yield of RNA extraction of fruit and leaf of A. deliciosa was ~2180.7?ng/µl (~545.175?µg/g?FW) and ~3424.9?ng/µl (~856.225?µg/g?FW), respectively with A260/A280 between 1.95 to 2.07 and A260/A230 higher than 2 indicating high RNA purity. While the averages yield of RNA extraction using previous methods from kiwifruit and leaf was 23?µg/g?FW and 527?µg/g?FW, respectively. Also, the average yields of genomic DNA from kiwifruit ranged from 52 to 98?ng/µl with A260/A230 between 0.60 to 1.64 and A260/A280 between 1.40 to 1.48. To our knowledge, this is the first report of a highly efficient and rapid method of RNA and DNA extraction in kiwifruit which can be used for a broad spectrum of the all fleshy fruits.

Total RNA Isolation from Planarian Tissues.

Isolation of high-quality and nondegraded RNA is a prerequisite for many modern molecular biology applications. A variety of RNA extraction products and protocols are available for the standard model organisms, yet species-specific peculiarities of less well studied organisms often require specific protocol adaptations. Here we describe a robust RNA extraction protocol for planarians that is widely used in the community. The protocol combines tissue homogenization in TRIzol with phenol-chloroform extraction and subsequent purification over commercial columns. The purified RNA can be used for many downstream applications, such as cDNA synthesis and next-generation sequencing (NGS, RNA-seq).

Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs.

Exosomes are small (30-150nm) vesicles of endosomal origin secreted by most cell types. Exosomes contain proteins, lipids, and RNA species including microRNA, mRNA, rRNA, and long noncoding RNAs. The mechanisms associated with exosome synthesis and cargo loading are still poorly understood. A role for exosomes in intercellular communication has been reported in physiological and pathological conditions both in vitro and in vivo. Previous studies have suggested that Schwann cell-derived exosomes regulate neuronal functions, but the mechanisms are still unclear. Here, we describe protocols to establish rat neonatal Schwann cell cultures and to isolate exosomes from the conditioned medium of these cultures by differential ultracentrifugation. To analyze the RNA content of Schwann cell-derived exosomes, we detail protocols for RNA extraction and next-generation sequencing using miRNA and mRNA libraries. The protocol also includes RNA sequencing of Schwann cells, which allows the comparison between RNA content from cells and the secreted exosomes. Identification of RNAs present in Schwann cell-derived exosomes is a valuable tool to understand novel roles of Schwann cells in neuronal function in health and disease.

Differential Gene Expression Analysis of Plants.

Since the next-generation sequencing (NGS) systems were invented and introduced to life science research about a decade ago, the NGS technology has extensively utilized in wide range of genomic, transcriptomic, and evolutionary studies. Compared with other eukaryotic species, the application of NGS technology in plant research reveals some challenges in sample preparation and data analysis due to some structural and physiological characteristics and genome complexity nature in plants. Hence, despite of the standard sample preparation and data process protocols widely used in high throughput transcriptomic analysis, we also describe the modified hot borate RNA extraction protocol specific for high quality and quantity plant total RNA isolation, and some comments and suggestions to achieve better assessments in the validation of RNA and library quality and data analysis.

The Use of High-Throughput Sequencing for the Study and Diagnosis of Plant Viruses and Viroids in Pollen.

This protocol details the wet lab preparation, extraction of fruit pollen samples, and analysis of the sequencing data following Illumina NextSeq small and total RNA sequencing. The protocol was developed for virus and viroid detection using NGS sequencing and was based on the results of a comparison between different extraction methods followed by yield, RNA purity, and integrity assessment. Moreover, the advantage of an additional ribosomal (r)RNA depletion step to the total RNA extraction protocol was evaluated. The smallRNA procedure is the preferred method of choice. If the total RNA protocol is chosen, the use of the mirVana kit followed by an rRNA depletion step is the best option. The library preparation and sequencing steps were outsourced. As a final step in the data analysis, the VirusDetect software was used to detect the viruses and viroids in the pollen samples.

Identificación de un gen codificante de polifenol oxidasa (PPO) en Theobroma cacao L. (cacao) de Ecuador

En Ecuador, las plantaciones de cacao presentan bajos promedios de producción debido a la diversidad de patógenos, especialmente a la infección por Moniliophthora roreri (monilia). Se cree que existe una relación entre el ataque del hongo y el aumento de los niveles de expresión de genes codificantes de polifenol oxidasas (PPOs) como mecanismo de defensa ante patógenos y herbívoros en diferentes plantas. Para la identificación de genes que codifican para PPOs, se seleccionaron hojas de cacao Nacional, provenientes de plantas resistentes y susceptibles a monilia, ubicadas en la Finca Experimental La Represa, propiedad de la Universidad Técnica Estatal de Quevedo. Se afinó un protocolo de extracción de ARN total de alta calidad para hojas de cacao recalcitrantes. Después de su retrotranscripción a ADNc, se realizaron ensayos de amplificación por PCR con diferentes primers, diseñados a partir de secuencias conservadas de PPOs. Los productos de amplificación permitieron la identificación de un gen de 961 pb, similar a un gen que codifica para la PPO predictiva de Theobroma cacao depositada en NCBI (XP_017978715.1) La identificación de este gen, es fundamental para evaluar a futuro los niveles de expresión y cuantificación en diferentes estados de desarrollo del fruto. Dicha cuantificación permitirá proponer herramientas de control para monilia y construir las bases para el mejoramiento genético del cacao Nacional.

Modifications in routine protocol of RNA isolation can improve quality of RNA purified from adipocytes

Adipose tissue is of interest in the context of its role in the pathogenesis of cardiovascular diseases. Modern experimental techniques require a well-purified RNA, but all the routine protocols for RNA extraction have a number of limitations in case of fatty tissues. Here we described a modified protocol for RNA extraction from human adipocytes based on routine column method. Suggested modifications optimized the sample preparation, lysis and washing lead to enhance RNA purity. We conclude that the current protocol for total RNA purification from adipocytes allows extracting a high-quality RNA devoid of fatty acids, organic solvents and salts contamination.

A fast and efficient protocol for small RNA extraction in Japanese plum and other Prunus species

Background: Small ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum ( Prunus domestica ) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV- Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry. Results: We describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV–P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159. Conclusion: We propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species. Normal 0 false false false ES-CL X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-ansi-language:ES-CL;} Background: Results: Conclusions:

Comparative assessment of four RNA extraction methods and modification to obtain high-quality RNA from Parthenium hysterophorus leaf

Isolation of high-quality RNA from weed plants such as Parthenium hysterophorus is a difficult task due to the hindrance caused by numerous secondary metabolites. Such metabolites not only affect the quality and yield of RNA, but also limit the quality of downstream applications. Therefore, the present study was undertaken to design a protocol for yielding RNA with better quality and quantity from P. hysterophorus leaf which could be suitable for functional genomics. To achieve the objective, four different important RNA extraction protocols, viz. acid guanidinium thiocyanate-phenol-chloroform, phenol-LiCl precipitation, TRIzol®, and PVP-ethanol were tested. The PVP-ethanol method proved to be best among the tested protocols. This method was further modified for obtaining improved quality and yield of RNA. The modified method successfully enhanced the yield of RNA from 280 to 334µgg-1 fresh weight. The absorbance ratio (A260/A280) was in the purity range of 1.9 that indicated the good quality of RNA. To prove the feasibility of the extracted RNA in PCR-based cDNA synthesis, actin transcripts were targeted and successfully amplified using suitable primers. The improved protocol thus not only improved the yield and quality of RNA, but also gave better results in reverse transcriptase PCR.

Elevation of brain-enriched miRNAs in cerebrospinal fluid of patients with acute ischemic stroke

BackgroundThe purpose of this study was to investigate the potential of cerebrospinal fluid miRNAs as diagnostic biomarkers of acute ischemic stroke using three different profiling techniques in order to identify and bypass any influence from technical variation.MethodsCerebrospinal fluid (CSF) from patients with acute ischemic stroke (n = 21) and controls (n = 21) was collected by lumbar puncture. miRNA analysis was performed with three different methods: 1) Trizol RNA extraction followed by Illumina Next Generation Sequencing (NGS) on all small RNAs, 2) Exiqon RNA extraction protocol and miRNA qPCR assays, and 3) validation of 24 selected miRNAs with Norgen Biotek RNA extraction protocol and Applied Biosystems qPCR assays.ResultsNGS detected 71 frequently expressed miRNAs in CSF of which brain-enriched miR-9-5p and miR-128-3p were significantly higher in CSF of stroke patients compared to controls. When dividing stroke patients into groups according to infarct size several brain-enriched miRNAs (miR-9-5p, miR-9-3p, miR-124-3p, and miR-128-3p) were elevated in patients with infarcts >2 cm3. This trend appeared in data from both NGS, qPCR (Exiqon), and qPCR (Applied Biosystems) but was only statistically significant in some of the measurement platforms.ConclusionsSeveral brain-enriched miRNAs are elevated in the CSF three days after stroke onset, suggesting that these miRNAs reflect the brain damage caused by ischemia. The expression differences seem, however, limited to patients with larger ischemic brain injury, which argues against the use of CSF miRNAs as diagnostic biomarkers of stroke based on current methods.

PRELIMINARY ANALYSIS FROM RNA-SEQUENCING DERIVED FROM FIVE BRAIN REGIONS IN BDR SAMPLES

The purpose of this study was to examine the quality of RNA that can be obtained from the Brains for Dementia Research (BDR) resource for RNA sequencing projects and to investigate the expression profiles obtained from different regions of the brain to provide insight into normal variation in gene expression across the brain. RNA was extracted from five brain regions from the same three samples obtained from the Brains for Dementia Research (BDR) repository using an optimised protocol to ensure high quality RNA for sequencing (n=15). RNA-sequence data from the Frontal, Temporal, Occipital, Parietal and Cerebellum regions of the brain were analysed for global differential gene expression. We developed a medium-throughput RNA extraction protocol for frozen brain samples which consistently produces high quantities of good quality RNA for sequencing. Data from our laboratory suggests an average RIN value of 5.6 is obtained from post-mortem brain tissue and more importantly DV200 values, now employed as a quality measurement for RNA-sequencing, are consistently over 70%, with an average of 87.5%. Preliminary findings suggest that whilst the Frontal, Temporal, Occipital and Parietal areas show similar gene expression profiles, the Cerebellum displays extensive differential gene expression above that expected by chance. The data obtained for such a study will complement the extensive serial clinical assessment information and detailed neuropathological data Available to researchers accessing the BDR resource.