High-quality Ribonucleic Acid Research Articles

Precise isolation of high-quality RNA from leaves and storage roots of cassava (Manihot esculenta Crantz) for gene expression studies

BackgroundThe isolation of Ribonucleic Acid (RNA) from leaves and storage roots of cassava (Manihot esculenta Crantz) is a challenging one, due to the presence of large amounts of polyphenolic compounds, polysaccharides, and tuber proteins. RNA with high quality and intact integrity is vital for gene expression studies. We hereby report a precise, reproducible, and less cumbersome technique for isolating high-quality RNA from leaves and storage roots of cassava with minimal contamination from polyphenols, polysaccharides, and other secondary metabolites, using affordable reagents. This protocol functions without guanidinium salts in the extraction buffer. The presence of guanidinium salts usually leads to the formation of agglomerates during the extraction of RNA from plant tissues with high starch contents.ResultsThe isolated RNA from leaves and storage roots of the ten cassava genotypes yielded between 1576.1 and 2861.9 µg/ml for RNA isolated from the leaf tissues and 2761.2–3873.5 µg/ml for RNA isolated from the storage roots. The A260:A280 ratios of the total RNA were more than 2.0 for both leaf and storage root samples, indicating minimal contamination from polysaccharides and polyphenols. The RNA samples recorded intact integrity, as demonstrated by clear 28 S and 18 S rRNA bands observed on agarose gel electrophoresis. The RNA integrity number (RIN) values ranged between 7.2 and 8.0. Also, the RNA samples were successfully used for transcriptome sequencing.ConclusionThe present method which yielded high-quality and transcriptionally competent RNA samples is suitable for use in gene expression studies and downstream applications in the molecular breeding of cassava and related root/tuber crops.

Optimized method for RNA extraction from leaves of forest tree species

Extraction of ribonucleic acid (RNA) from woody plants is a difficult task due to the peculiarities of plant material rich in polysaccharides and starch. The available techniques are often ineffective, since they result in the absence/reduced quality/reduced amount of RNA in the final preparation. The method we have optimized is based on the use of cethyltrimethyl ammonium bromide (CTAB), purification by phenol-chloroform extraction, use of lithium chloride and ammonium acetate. The method showed high efficiency for the extraction of RNA from the leaves of birch and poplar samples, in vitro and mature plants, in comparison with previously used methods (extraction using NucleoSpin® RNA Plant (Macherey-Nagel, Germany) columns, Su (2009) method, standard guanidine thiocyanate method). Electropherograms of RNA preparations showed its high integrity and concentration (up to 85 ng/μl), significantly higher purity of the preparation (up to 2.7 times). Purification of the preparation in the process of extraction can significantly reduce the yield of desoxyribonucleic acid (DNA). The optimized method is highly reproducible and can be used for further research, complementary DNA (cDNA) synthesis, qualitative and quantitative PCR analysis. The method allows obtaining high-quality RNA from other objects of agricultural and forest plants.

Magnetic Ionic Liquids as Solvents for RNA Extraction and Preservation.

Ribonucleic acid (RNA) is particularly sensitive to enzymatic degradation by endonucleases prior to sample analysis. In-field preservation has been a challenge for RNA sample preparation. Very recently, hydrophobic magnetic ionic liquids (MIL) have shown significant promise in the area of RNA extraction. In this study, MILs were synthesized and employed as solvents for the extraction and preservation of RNA in aqueous solution. RNA samples obtained from yeast cells were extracted and preserved by the trihexyl(tetradecyl) phosphonium tris(hexafluoroacetylaceto)cobaltate(II) ([P66614+][Co(hfacac)3–]) and trihexyl(tetradecyl) phosphonium tris(phenyltrifluoroacetylaceto)cobaltate(II) ([P66614+][Co(Phtfacac)3–]) MIL with a dispersion of the supporting media, polypropylene glycol, at room temperature for up to a 7 and 15 day period, respectively. High-quality RNA treated with ribonuclease A (RNase A) was recovered from the tetra(1-octylimidazole)cobaltate(II) di(l-glutamate) ([Co(OIM)42+][Glu–]2) and tetra(1-octylimidazole)cobaltate(II) di(l-aspartate) ([Co(OIM)42+][Asp–]2) MILs after a 24 h period at room temperature. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and agarose gel electrophoresis were used to determine the effect of RNA preservation. Furthermore, the preservation mechanism was investigated by exploring the partitioning of RNase A into the MIL using high-performance liquid chromatography.

Modified CTAB protocol for RNA extraction from Lemon balm (Melissa officinalis L.)

Ribonucleic acid (RNA) quality and integrity are crucial for many studies in plant molecular biology. High-quality RNA extraction from plants with high levels of compounds such as polysaccharides, polyphenols, and other secondary metabolites are problematic. RNA extraction from Lemon balm tissues can be difficult due to the presence of polyphenolic and polysaccharide compounds or can be done by expensive protocols. This study shows improvement of a CTAB-based protocol which allows rapid and easy isolation of high-quality RNA from Lemon balm plant. The RNA obtained is suitable for cDNA synthesis and RT-PCR experiments.

High-quality total RNA extraction from Magnolia sieboldii K. Koch seeds: a comparative evaluation

Magnolia sieboldii K. Koch seed is characterized with having deep dormancy. The inner molecular regulation mechanism has not been investigated because of the absence of a protocol for total RNA (ribonucleic acid) extraction. The extraction of high-quality RNA is important and can be a limiting factor in plant molecular biology experiments. Sufficient high-quality total RNA is required to elucidate the molecular regulation mechanism of germination. However, M. sieboldii seeds with large amounts of secondary metabolites also contain recalcitrant tissues for RNA isolation. We found two simple and low-cost RNA extraction methods for M. sieboldii seeds by evaluating and selecting eight types of methods and further optimizing these methods. The two methods were not only suitable for extracting M. sieboldii seed RNA but also applicable to RNAs from several other tissues. Total RNA extracted through these approaches was applicable for general molecular biology experiments such as qRT-PCR (quantitative real-time polymerase chain reactions). The protocols also meet the strict harsh requirements for transcriptome sequencing and small RNA sequencing. This study provides a powerful approach for future studies at the transcription level.

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA.

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells.

Isolation of total ribonucleic acid from fresh and frozen-thawed boar semen and its relevance in transcriptome studies

The main objective of this study was to isolate high-quality total ribonucleic acid (RNA) from raw fresh semen and frozen-thawed boar semen, using a protocol comprising the conventional TRIzol assay and a membrane-based technique, the PureLink RNA mini kit. Bioanalyzer profile revealed that the sperm RNA size distributions comprised mainly intact RNA ranging from 1500 to 1800 bp, without any detectable residual genomic deoxyribonucleic acid (DNA) or 28S ribosomal RNA (rRNA). Spectrophotometric quantifications of the total RNA yielded 1.64 to 2.44 μg/106 spermatozoa, irrespective of the sperm source. The TRIzol/PureLink protocol allowed the isolation of high-quality intact RNA from boar spermatozoa, which is required for transcriptome analysis on high-throughput RNA-sequencing (RNA-Seq) data. Such an approach is relevant to identifying sperm messenger RNA (mRNA transcripts) that are associated with boar semen freezability.Keywords: cryopreservation, RNA-Seq, semen quality

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis.

ObjectivesStudies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues.Materials and MethodsTissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR.ResultsNo differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility.ConclusionFor the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3.

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice.

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

PRESERVING LYMPHOCYTE RIBONUCLEIC ACID FOR ANALYSIS AND TRANSPORT FROM WHOLE BLOOD IN RURAL AREAS.

Background Preservation of human ribonucleic acid (RNA) from whole blood samples in rural areas with very few resources has previously been very difficult and/or expensive. This has limited clinical research trials in third world countries and distant rural areas in developed countries. The major drawback to being able to study these populations is the difficulty in transporting blood samples while maintaining the integrity and preventing degradation of the lymphocytes, DNA, protein, and RNA. Of these, RNA is the most difficult and expensive to preserve. Objective To develop an inexpensive, simple, and reliable method to collect and preserve high-quality RNA from whole blood lymphocytes for prolonged transport. Methods Blood was collected from young adult rats and treated four different ways. For the first method, blood was placed in an equal volume of RNAlater® and frozen in dry ice and then placed in −80°C for 1 week. This was then thawed on ice and processed. The second method used an equal volume of RNAlater® but was not frozen. The third method was diluted with an equal volume of water before processing. In the fourth method, fresh, undiluted blood was used. RNA was isolated with Trizol® LS immediately after collection or thawing. cDNA was then synthesized from this RNA. An RT-PCR was then run using the housekeeping gene GAPDH. Results The sample that had the highest expression was the untreated and undiluted whole blood sample at an average of 22.57 Ct. The second highest expression was the blood that was treated with RNAlater® and frozen for 1 week. This expressed at an average of 25.03 or 2.5 cycles later. The next highest expression was the blood diluted with water. The Ct value for it was an average of 30.89 or 8 cycles after the untreated blood. The fourth sample expressed at an average of 32.92 or over 10 cycles beyond the untreated blood. Conclusion We have successfully developed a simple, inexpensive, and reliable method of collection and stabilization of high-quality RNA from whole blood lymphocytes for transport and later experimental use. This preservation of RNA, when compared with fresh blood treated with RNAlater® or diluted with water, is expressed almost as highly as untreated or undiluted blood. We speculate that this procedure will greatly enhance the ability to study lymphocyte RNA in third world and rural area populations.

Optimization of RNA yield, purity and mRNA copy number by treatment of urine cell pellets with RNA later

Background: We have shown that measurement of mRNA for cytotoxic attack proteins perforin and granzyme B in urinary cells is a noninvasive means of diagnosing acute rejection of human renal allografts. Urinary cell mRNA studies have yielded useful information in other patient populations such as patients with cancer. The isolation of sufficient and high quality ribonucleic acid (RNA) from urinary cells however is problematic. RNA later, an RNA stabilization solution, has been reported to optimize RNA isolation from tumor tissues stored at room temperature and from pigment-rich ocular tissues. Methods: We explored whether the addition of RNA later to urine cell pellets improves RNA yield, enhances purity and facilitates measurement of low abundance mRNAs. We measured, with the use of real-time quantitative polymerase chain reaction (PCR) assay, levels of expression of a constitutively expressed gene 18S rRNA and mRNA for granzyme B and transforming growth factor-β 1 (TGF-β 1) in urine specimens and renal biopsies obtained from renal allograft recipients. Results: RNA yield ( P<0.01, Wilcoxon signed rank test) and the A260/ A280 ratio ( P<0.01) were both higher with urine cell pellets treated with RNA later prior to snap freezing compared to cell pellets that were not treated with RNA later prior to snap freezing. Levels (copy number per 1 μg of total RNA) of 18S rRNA ( P<0.02), granzyme B mRNA ( P=0.002) and TGF-β 1 ( P=0.02) were all higher with treated urine cell pellets compared to untreated cell pellets. Kruskall–Wallis one way analysis of variance and pair-wise comparisons with Student–Newman–Keuls test showed that the levels of mRNA for granzyme B ( P<0.05) and TGF-β 1 ( P<0.05) are significantly different between renal allograft biopsies and untreated urine cell pellets but not between the biopsy specimens and RNA later-treated urine cell pellets. Conclusions: The addition of RNA later to urine cell pellets improves RNA isolation from urinary cells and facilitates measurement of low abundance mRNAs.

A method for the extraction of high-quality RNA and protein from single small samples of arteries and veins preserved in RNAlater

Introduction: The mesenteric vasculature plays an important role in cardiovascular homeostasis, both in terms of arterial resistance and venous capacitance. However, the limited quantity of tissue available and the activity of RNases and proteases during dissection of the vessels make it difficult to study gene and protein expression in this tissue. Methods: The mesenteric arcade was harvested from rats. The mesenteric arteries and veins were separated from each other and from contaminating adipose, nerve, and connective tissue by blunt dissection while submerged in RNAlater (Ambion). Total ribonucleic acid (RNA) and protein were extracted from the same mesenteric artery and vein samples. Results: Good quality RNA and protein were obtained by this method. The method preserved the content of proteins including biglycan, adrenergic receptors, estrogen receptors, and protein kinase Cδ, and also preserved the differential expression of estrogen receptors. Discussion: A method for the isolation of high-quality RNA and protein from the arteries and veins of the same mesenteric sample is described. The content of proteins from a variety of cellular compartments and molecular weights was preserved by this method, as well as differential expression of proteins. This method will facilitate the study of gene and protein expression in the mesenteric vasculature in response to pharmacologic treatments and in disease states.