Isolation Of Ribonucleic Acid Research Articles

A comparison of five methods to maximize RNA and DNA isolation yield from adipose tissue.

Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn't allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization.

Chemokine CXCL8 and its receptors as markers of colorectal cancer

BACKGROUND: The chemokine CXCL8 and its receptor are involved in the activation and transport of inflammatory mediators and regulate the proliferation and renewal of cancer stem cells in colon cancer. It is believed that CXC signaling may be associated with poor prognosis in colorectal cancer.
 AIM: To study the possibilities of using CXCL8-CXCR1/2 pathway indicators as markers in colorectal cancer.
 MATERIAL AND METHODS: Isolation of ribonucleic acid (RNA) from histological sections of tumors obtained intraoperatively from 59 patients diagnosed with colorectal cancer was carried out using magnetic particles, and quantitative polymerase chain reaction in real time was performed. Calculation of the normalized expression of the CXCL8 and CXCR1 genes relative to the referee gene was done using special software. Statistical data processing was performed using Statistica 3.0, BioStat v. 7.1 programs. Comparisons of characteristics were performed using the Mann–Whitney U test. Cox and Kaplan–Meier criteria were used to analyze overall and disease-free survival.
 RESULTS: The expression of CXCL8 in intestinal adenocarcinoma cells with low differentiation [Me (Q1–Q3) — 8.770 (1.127–1.114)] was significantly higher than in the groups of moderately and well differentiated tumors (p=0.004 and p=0.012, respectively); and in tumor tissue refractory to chemotherapy, it was significantly higher [4.374 (2.052–7.045)] compared to resistant [2.200 (1.388–5.037); p=0.008] and sensitive [1.624 (0.739–2.586); p=0.042]. The level of CXCR1 messenger RNA in tumor tissue was increased in the presence of BRAF mutation [3.645 (0.801–1.090); p=0.009] and low tumor differentiation [6.965 (3.938–12.225); p=0.002], as well as in tumor tissue refractory to FOLFOX/XELOX chemotherapy [46.224 (27.580–83.570); p=0.0009].
 CONCLUSION: Expression of components of the CXCL8-CXCR1/2 signaling pathway in tumor tissue may be a marker of sensitivity to FOLFOX/XELOX chemotherapy in colorectal cancer.

Investigation of the effect of belinostat on MCF-7 breast cancer stem cells via the Wnt, Notch, and Hedgehog signaling pathway.

To evaluate belinostat's (PXD101) activity on MCF-7 breast cancer stem cells (CSCs) via Wnt, Notch, and Hedgehog. This research study was carried out at the Department of Medical Biology, Necmettin Erbakan University, Konya, Turkey, from June 2017 to July 2019. The effect of PXD101 on MCF-7 cell viability was determined by cell proliferation kit (XTT). Following belinostat treatment, CD44+/CD24- MCF-7 CSCs were isolated by FACS. Ribonucleic acid isolation and copy-deoxyribonucleic acid synthesis were carried out using HEK-293 cells, MCF-7 cells, and MCF-7 CSCs. Expression changes of metastasis-related genes, Wnt, Hedgehog, Notch, and stem cell markers were analysed by quantitative polymerase chain reaction. The IC50 in MCF-7 cancer cells was 5 μM for 48 hours. The FACS analysis indicated that 2% of the MCF-7 cancer cells were CSCs. Following belinostat treatment, the MCF-7 cell count decreased by 44%, and the MCF-7 CD44+/CD24- CSC count decreased by 66%. Belinostat treatment reduced the expression of metastasis, Wnt, Notch, Hedgehog, and stem cell marker genes. Belinostat has a potential effect on the differentiation and self-renewal of breast CSCs.

RNA Isolation Method in Marginal Crops with High Agronomic Potential.

Ribonucleic Acid (RNA) isolation is a basic technique in the field of molecular biology. The purpose of RNA isolation is to acquire pure and complete RNA that can be used to evaluate gene expression. Many methods can be used to perform RNA isolation, all of them based on the chemical properties of nucleic acids. However, some of them do not achieve high RNA yields and purity levels when used in a number of marginally studied crops of agronomic importance, such as grain and vegetable amaranth plants. In the method described here, the use of guanidinium thiocyanate and two additional precipitation steps with different reagents designed to obtain high yields and RNA purity levels from diverse plant species employed for plant functional genomics studies is described.

Ribonucleic acid isolation from human mononuclear cell culture with magnetic beads pre-enrichment for molecular analysis

Ribonucleic acid isolation from human mononuclear cell culture with magnetic beads pre-enrichment for molecular analysis

Room temperature roll-to-roll additive manufacturing of polydimethylsiloxane-based centrifugal microfluidic device for on-site isolation of ribonucleic acid from whole blood

Polymer-based lab-on-a-disc (LoaD) devices for isolating ribonucleic acid (RNA) from whole blood samples have gained considerable attention for accurate biomedical analysis and point-of-care diagnostics. However, the mass production of these devices remains challenging in manufacturing cost and sustainability, primarily due to the utilization of a laser cutter or router computer numerical control (CNC) machine for engraving and cutting plastics in the conventional prototyping process. Herein, we reported the first energy-efficient room-temperature printing-imprinting integrated roll-to-roll manufacturing platform for mass production of a polydimethylsiloxane (PDMS)-based LoaD to on-site isolate ribonucleic acid (RNA) from undiluted blood samples. We significantly reduced energy consumption and eliminated thermal expansion variations between the mold, substrate, and resists by accelerating the PDMS curing time to less than 10 min at room temperature without using heat or ultraviolet radiation. The additive manufacturing technology was applied to fabricate a multi-depth flexible polymer mold that integrated macro (2 mm) and micro-sized (500 μm) features, which overcomes the economic and environmental challenges of conventional molding techniques. Our integrated R2R platform was enabled to print adhesion-promoting films at the first printing unit and continuously in-line imprint with a high replication accuracy (99%) for high-volume manufacturing of a new centrifugal microfluidic chip with an enhancement of mixing performance by integrating an efficient mixing chamber and serpentine micromixer. This research paved the way for scalable green manufacturing of large-volume polymer-based microfluidic devices, often required in real-world sample-driven analytical systems for clinical bioanalysis.

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.

Approaches to the use of stem cells in regenerative medicine

PurposeThe goal of regenerative medicine is to replace or restore missing, dysfunctional, or damaged cells, tissues and organs of a person to reproduce their normal function. The main approaches are cell therapy, tissue engineering, and gene therapy. Postnatal neural crest-derived multipotent stem cells (NC-MSC) are a promising cell type for use in regenerative medicine. This is due to the specific features of their embryonic origin and the role of the neural crest in phylogeny and ontogeny of vertebrates. MethodsThe study used research in vitro culture (monolayer cell culture, 3D culture based on hydrogels, organotypic culture of hippocampal slices, spherogenesis, directed differentiation); flow cytometry; cytochemical, immune-cytochemical and histomorphometric analysis; molecular genetic (RNA (ribonucleic acid) isolation, PCR (polymerase chain reaction) with reverse transcription, real-time PCR, nucleic acid electrophoresis); microscopy (transmitted light, phase contrast, fluorescent, confocal laser scanning); microsurgical; statistical analysis. ResultsIn this systematic review, the results showed that recently the neural crest-derived cells have been isolated from a wide range of tissues and organs of mammals at the postnatal stage of development. These cells, at least in vitro, demonstrate the ability to self-repair and multilinear differentiation into neurons, Schwann cells, melanocytes, adipocytes, osteoblasts, chondrocytes, and other types of cells, that is, according to their functional characteristics, they are multipotent stem cells. ConclusionAccording to the obtained results, tissue sources of postnatal neural crest-derived multipotent stem cells differ considerably in the degree of invasiveness of biopsy sampling, as well as the possibility of obtaining a homogeneous population of NC-MSCs, which is important for further clinical use.

Optimization of RNA storage in a biobank, as well as methods for manual and automated isolation of RNA from whole blood and leukocyte fraction

Aim. To optimize the technique for the isolation and storage of ribonucleic acid (RNA) from whole blood and leukocyte fraction.Materials and methods. Comparison of isolation quality was carried out for RNA samples obtained from 228 leukocyte samples and 198 whole blood samples. Isolation was performed from fresh and frozen samples using ExtractRNA™ reagent and a MagNA Pure Compact automated system. Various methods of removing erythrocytes (centrifugation and treatment with hemolytic agents from two manufacturers) were tested, as well as freezing with and without preservatives for subsequent RNA isolation.Results. Twenty-one combinations of conditions were tested. The highest quality RNA was isolated by manual extraction using the ExtractRNA™ reagent from a fresh leukocyte fraction, purified by the Amplisens hemolytic agent (successful extraction — 94%, median RIN=8,4); frozen in IntactRNA™, purified by leukocyte fraction centrifugation (successful extraction — 100%, median RIN=8); frozen in ExtractRNA™, purified by leukocyte fraction centrifugation (successful extraction — 100%, median RIN=9,3).Conclusion. RNA can be isolated from frozen blood fractions, which is not inferior in quality to that isolated from fresh samples. Thus, it is not necessary to isolate RNA immediately after the receipt of biological material.

Performance comparison of commercial RNA extraction kits for the diagnosis of SARS-CoV-2: A laboratory perspective

Introduction: The COVID-19 pandemic has resulted in an increased need for molecular diagnostics. Ribonucleic acid (RNA) extraction is a crucial step in the detection of severe acute respiratory syndrome coronavirus-2 (SARS-COV-2). The resulting outcome depends on the performance of the RNA extraction kit used.Methods: This study evaluated the efficiency of four different commercial RNA extraction kits for the detection of SARS-COV 2. The reproducibility and performance efficiency of each kit were assessed and compared. The performance, processing time and storage conditions were also considered in the evaluation.Results: A slightly lesser cycle threshold (Ct) values were observed in RNA extracted using HiPurA Viral RNA Purification Kit. Invitrogen PureLink RNA Mini Kit was found to be time-saving and user-friendly.Conclusions: All the RNA isolation kit yielded the best results; however, if time is the important considerations, Invitrogen PureLink RNA Mini Kit has the shortest processing time.

An improved procedure for the isolation of Ribonucleic acid from methicillin-resistant Staphylococcus aureus

Extraction and purification of ribonucleic acid (RNA) from Gram-positive methicillin resistant Staphylococcus aureus (MRSA) is problematic, because the MRSA has a rigid cell wall that contains lipoteichoic acid and peptidoglycan, thus causing difficulty when utilizing the standard methods. For this reason, the aim of the current study was to improve and modify the method of extraction of RNA from MRSA, with good integrity, purity, low cost, and with saved time of extraction. A fast and an inexpensive method involving the use of acid phenol: chloroform (5: 1 [v/v]) at low pH (4.5), with lysostaphin and Triton X-100 for effective isolation of RNA from the MRSA is developed. As a result of this study, yields of this method presented high concentration of RNA 1175.26 ng/ µl/ 3 ml of bacterial culture broth, with high RNA integration number (RIN). In similar assays such as using; the RNeasy Mini kit, GeneJET RNA purification kit, TRIzol kit and hot phenol: chloroform (1: 1 [v/v]) extraction method, they yielded low concentrations of RNA (92-700 ng/ µl); with lower purity, quantity, and also little integrity, compared to using the current acid phenol chloroform (5: 1 [v/v]) extraction method. In conclusion, this new method for extraction of RNA from MRSA can be used to save time, cost, and provide high quality of RNA.

Human Sperm Cells After Purification Using SCLB Can Be Stored at 4o, -20o, or -80oC Before Small RNA Isolation

There have been many studies about pre-analysis for sperm RNA examination which compared sperm purificationmethods, RNA isolation methods, sequencing methods, and semen storage before analysis. However, there is a lack ofstudies that determine the ideal storage temperature after sperm cell purification before RNA analysis, especially small RNAanalysis. The aim of this study was to determine the preferred storage temperature for human sperm cells after spermpurification using Somatic Cell Lysis Buffer (SCLB) before sperm small ribonucleic acid (RNA) isolation and analysis. Thisstudy was a true laboratory experiment using the post-test only control group design. The samples were 13 fresh humansemen that has been purified using SCLB. The sperm cells were then diluted and divided into four aliquots with differenttreatments. The first aliquot that served as a control group was immediately purified while the other three aliquots were0 0 0 stored for seven days at different temperatures as follows: 4 C, -20 , and -80 C. After the small RNA isolation, RNA levelbetween each group was compared. Micro volume spectrophotometer measured RNA level. The median of small RNA6 yields of the control group was 49.8 (5.33-522.46) ng/10 sperm cells. There was no significant difference in median of smallRNA yields of the control group and that of other groups. The median of the other groups with storage temperature0 0 0 6 of 4 C, -20 , and -80 C was 41.09 (7.03-1448.31), 65.95 (7.99-301.16), and 76.42 (10.45-434.25) ng/10 sperm cells,respectively (p-value= 0.314; α=5%). This condition suggested that after purification using SCLB, human sperm cells can be0 0 0 stored at temperatures of 4 C, -20 , or -80 C for seven days, depending on each laboratory facility.

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.

Evaluation of gene expression levels in the diagnosis of lung adenocarcinoma and malignant pleural mesothelioma.

This study aims to evaluate gene expression levels in the diagnosis of lung adenocarcinoma and malignant pleural mesothelioma both which have a distinct treatment and prognosis. Between January 2012 and January 2014, 12 newly diagnosed patients with a lung adenocarcinoma, 12 patients with malignant pleural mesothelioma, and eight healthy individuals as the control group were included. After treatment of the fresh samples of lung adenocarcinoma stored at -80°C for ribonucleic acid isolation, and paraffin-embedded tissues of patients with malignant pleural mesothelioma were deparaffinized, complementary deoxyribonucleic acid synthesis and expression of 84 genes associated with deoxyribonucleic acid repair were analyzed via real-time polymerase chain reaction assay. According to the expression of tumor cells, expression of each fold change was calculated. The BRCA1, BRCA2, CDK7, MLH3, MSH4, NEIL3, SMUG1, UNG, XRCC2, and XRCC4 genes showed more than five-fold higher expression in the patients with lung adenocarcinomas, compared to the control group. The patients with malignant pleural mesothelioma showed a five-fold higher expression in the APEX2, BRCA1, BRCA2, CDK7, MLH1, MLH3, MSH3, MSH4, NEIL3, PARP2, PARP3, PMS1, RAD50, RAD51, RAD51B, RAD51D, RAD52, RPA3, SMUG1, UNG, XPA, XRCC2, and XRCC4 genes, compared to the control group. Comparing malignant pleural mesothelioma with lung adenocarcinoma cases, we found that CDK7, MLH1, TREX1, PRKDC, XPA, PMS1, UNG, and RPA3 genes were overexpressed. Our study results showed differences between expression profiles of deoxyribonucleic acid repair genes in lung adenocarcinoma and malignant pleural mesothelioma cells. Based on our study results, we suggest that TREX1, PRKDC, and PMS1 genes may play a key role in the differential diagnosis of these two entities.

Gene Expression Profiles in Children With Suspected Sepsis

Sepsis recognition is a clinical challenge in children. We aim to determine whether peripheral blood gene expression profiles are associated with pathogen type and sepsis severity in children with suspected sepsis. This was a prospective pilot observational study in a tertiary pediatric emergency department with a convenience sample of children enrolled. Participants were older than 56 days and younger than 18 years, had suspected sepsis, and had not received broad-spectrum antibiotics in the previous 4 hours. Primary outcome was source pathogen, defined as confirmed bacterial source from sterile body fluid or confirmed viral source. Secondary outcome was sepsis severity, defined as maximum therapy required for shock reversal in the first 3 hospital days. We drew peripheral blood for ribonucleic acid isolation at the sepsis protocol activation, obtained gene expression measures with the GeneChip Human Gene 2.0 ST Array, and conducted differential expression analysis. We collected ribonucleic acid samples from a convenience sample of 122 children with suspected sepsis and 12 healthy controls. We compared the 66 children (54%) with confirmed bacterial or viral infection and found 558 differentially expressed genes, many related to interferon signaling or viral immunity. We did not find statistically significant gene expression differences in patients according to sepsis severity. The study demonstrates feasibility of evaluating gene expression profiling data in children evaluated for sepsis in the pediatric emergency department setting. Our results suggest that gene expression profiling may facilitate identification of source pathogen in children with suspected sepsis, which could ultimately lead to improved tailoring of sepsis treatment and antimicrobial stewardship.

Isolation of RNA and beta-NAD by phenylboronic acid functionalized, monodisperse-porous silica microspheres as sorbent in batch and microfluidic boronate affinity systems.

Monodisperse-porous silica microspheres 5.5 μm in size were obtained by a staged shape templated hydrolysis-condensation method, with a bimodal pore-size distribution. 3-aminophenylboronic acid (APBA) was covalently attached onto the silica microspheres with a capacity of 0.476 mmol APBA/g microspheres. The boronate affinity isolation behaviour of ribonucleic acid (RNA) containing cis-diol at 3'-end was investigated by using APBA attached-silica microspheres as the sorbent in batch fashion. A short-chain diol carrying agent, β-nicotinamide adenine dinucleotide (β-NAD) was used as a target molecule with stronger affinity for phenylboronic acid ligand. The maximum equilibrium adsorptions for RNA and β-NAD were determined as 60 and 159 mg/g sorbent, respectively. By using the synthesized sorbent, phosphate buffer at pH 7.0 containing sorbitol was successfuly used as a mild elution medium for obtaining quantitative desorptions with both RNA and β-NAD. RNA isolations from mammalian and bacterial cells were successfully performed while protecting the structural integrity of RNA via boronate affinity interaction in batch fashion. A microfluidic boronate affinity system including a microcolumn 300 μm in diameter was also constructed using APBA attached-silica microspheres as the stationary phase. The breakthrough curves of microfluidic system were obtained by studying with different feed concentrations of RNA and β-NAD. Quantitative desorptions and satisfactory isolation yields were obtained with RNA and β-NAD in the microfluidic system. The proposed system is useful for boronate affinity applications in genomics or proteomics in which valuable cis-diols at low concentrations are recovered from low-volume samples.

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.

Rapid and Efficient Method of Total RNA Isolation from Milk Fat for Transcriptome Analysis of Mammary Gland

Isolation of good quality ribonucleic acid (RNA) from mammary glands of elite animals is often hindered by invasive method of mammary tissue sampling, ethical permission, time consuming, and repeated biopsies from the same animal at different time points. The aim of the present study was to optimize a protocol for RNA isolation from milk fat that is suitable for transcriptome analysis. In this study, authors isolated good quality RNA from milk fat of goats and buffalo milk by combining two methods namely Trizol and GenElute mammalian RNA isolation kit. The results showed good quality as well as high quantity of total RNA suitable for downstream applications like reverse transcriptase-quantitative polymerase chain reaction and next generation sequencing.

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