RNA Purity Research Articles

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omittedsize selection, to allow capture of more RNA species.

Optimised isolation method for RNA extraction suitable for RNA sequencing from feline teeth collected in a clinical setting and at post mortem

Advanced next generation sequencing approaches have started to reveal the cellular and molecular complexity of the microenvironment in many tissues. It is challenging to obtain high quality RNA from mineralised tissues. We developed an optimised method of RNA extraction from feline teeth collected in a clinical setting and at post mortem. Teeth were homogenised in phenol-guanidinium solution at near-freezing temperatures and followed by solid-phase nucleic acid extraction utilising a commercially available kit. This method produced good RNA yields and improved RNA quality based on RNA integrity numbers equivalent (RINe) from an average of 3.6 to 5.6. No correlation was found between RNA purity parameters measured by A260:280 or A230:260 ratios and degree of RNA degradation. This implies that RNA purity indicators cannot be reliably used as parameters of RNA integrity. Two reference genes (GAPDH, RPS19) showed significant changes in expression levels by qPCR at low and moderate RINe values, while RPL17 was stable at all RINe values tested. Furthermore, we investigated the effect of quantity and quality of RNA on the quality of the resultant RNA sequencing (RNA-Seq) data. Thirteen RNA-seq data showed similar duplication and mapping rates (94 to 95%) against the feline genome regardless of RINe values. However one low yield sample with a high RINe value showed a high duplication rate and it was an outlier on the RNA-seq multidimensional scaling plot. We conclude that the overall yield of RNA was more important than quality of RNA for RNA-seq quality control. These results will guide researchers who wish to perform RNA extractions from mineralised tissues, especially if collecting in a clinical setting with the recognised restraints that this imposes.

RNA Purity, Real-Time PCR Sensitivity, and Colon Segment Influence mRNA Relative Expression in Murine Dextran Sodium Sulfate Experimental Colitis.

The dextran sodium sulfate (DSS) model of colitis is widely used as a result of its simplicity and reproducibility and because it mimics clinicopathological disease features. Its effectiveness depends on the mouse strain, DSS MW, and brand. Quantitative RT-PCR (qRT-PCR) is highly sensitive for analyzing cytokine mRNA expression. We analyzed an acute model of DSS treatment in Balb/c mice for the onset of colitis using qRT-PCR for the quantification of a mouse cytokine transcript. We compared differences among 1--and 2-step qRT-PCR for transcript quantification, the effect of multiple concentrations of DSS, and the use of 2 reference genes in 3 portions of the colon. A reliable and sensitive 1-step protocol for qRT-PCR was established with a modified double LiCl precipitation for RNA isolation. The variability of 2 reference genes, β-actin and eukaryotic elongation factor 2, was compared, and expression of IL-6 was analyzed in 3 segments of the colon. The RNA cleaning protocol prevented inhibition of qRT-PCR by DSS, and RNA loss was minimized. No clinical differences among the different DSS concentrations were seen on d 7, but higher concentrations resulted in the appearance of earlier symptoms. Higher efficiency and sensitivity of the 1-step qRT-PCR reaction using eukaryotic elongation factor 2 were obtained and also less variability. Although expression levels of IL-6 were high in the middle and distal colon, the middle section had consistently less variability in values. Thus, this segment is recommended for future studies. These factors influence the statistical significance of data and need to be considered to get accurate and reliable results and to improve comparisons of the published colitis experiments.

Isolation of high-quality RNA from intervertebral disc tissue via pronase predigestion and tissue pulverization.

The isolation of high‐quality RNA from the intervertebral disc and especially from the nucleus pulposus is challenging due to the low cellularity and high proteoglycan content of this tissue. In this study, we report a simple modification of the standard guanidinium thiocyanate‐phenol‐chloroform extraction method, which involves enzymatic predigestion of the tissue prior to standard RNA isolation. Yield, purity and integrity of RNA isolated from bovine nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus were compared among complete matrix digestion, predigestion and pulverization, pulverization alone, and pulverization followed by on‐column purification. With predigestion, the average yield of RNA obtained from bovine nucleus pulposus was 8.82 ± 2.05 ng/mg of wet tissue with 260/280 and 260/230 optical density ratios of 1.91 ± 0.15 and 1.84 ± 0.30, respectively. RIN analysis indicated that RNA quality was best preserved with the predigestion method (RNA integrity number > 7), and the extracted RNA was suitable for real‐time polymerase chain reaction. This method is of importance for gene expression studies on intervertebral disc development, degeneration and repair, and we anticipate that it may be further applied to other tissues rich in proteoglycans.

Optimized Storage Methods of RNA Extraction from Formalin Fixed Paraffin Embedded Tissue

Background: RNA extraction from Formalin Fixed Paraffin Embedded tissue (FFPE) provides valuable information. The main obstacle for pure RNA extraction from FFPE specimens is RNA degradation over time and low yield of RNA due to chemical processing. In the present study, RNA extraction from FFPE specimens were optimized for storage time, proteinase K concentration, and tissue size hemogenation. Methods: To evaluate the effect of storage time on RNA extraction yield, total RNA from 78 FFPE breast tissue specimens (1 - 3 years n = 52, and less than one year, n = 26) were extracted by High Pure Paraffin Kit (Roche). The effect of 2 different proteinase K on RNA was evaluated by proteinase K, and the effect of homogenization was evaluated using 2 different section sizes (10 µm and 5X2 µm). Extracted RNA was converted to cDNA. The SYBER Green Real time PCR was performed for quantitative analysis using ABI7900 Real time PCR. Results: The results indicated that FFPE storage time affected the yield of RNA extraction. The more the time of storage, the less RNA could be obtained (r = -0.38, P = 0.01). Smaller tissue section size seems to increase the amount of efficient RNA extraction from FFPE, probably through appropriate tissue lysis and more RNA release. According to the current study, proteinase K (Endopeptidase K) from different companies also affected on the quality of RNA extracted from FFPE (P = 0.032). Conclusion: Different optimization strategies enhance quality, purity, and quantity of RNA extracted from FFPE, which is critical in gene expression studies, like qRT-PCR.

Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples

BackgroundGlomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartment-specific glomerular gene expression analysis of formalin-fixed paraffin-embedded renal biopsies could improve research applications. The major challenge for such studies is to obtain good-quality RNA from small amounts of starting material, as applicable for the analysis of glomerular compartments. In this work, we provide data and recommendations for an optimized workflow of glomerular mRNA analysis.ResultsWith a proper resolution of the camera and screen provided by the next generation of micro-dissection systems, we are able to separate parietal epithelial cells from glomerular tufts. Selected compartment-specific transcripts (WT1 and GLEPP1 for glomerular tuft as well as PAX2 for parietal epithelial cells) seem to be reliable discriminators for these micro-dissected glomerular substructures. Using the phenol–chloroform extraction and hemalaun-stained sections (2 µm), high amounts of Bowman’s capsule transections (> 300) reveal sufficient RNA concentrations (> 300 ng mRNA) for further analysis. For comparison, in unstained sections from a number of 60 glomerular transections upwards, a minimum amount of 157 ng mRNA with a reasonable mRNA purity [A260/A280 ratio of 1.5 (1.4/1.7) median (25th/75th percentiles)] was reversely transcribed into cDNA. Comparing the effect of input RNA (20, 60, 150 and 300 micro-dissected glomerular transections), transcript expression of POLR2A significantly correlated when 60 and 150 laser micro-dissected glomerular transections were used for analysis. There was a lower inter-assay coefficient of variability for ADAMTS13, when at least 60 glomerular transections were used. According to the algorithms of geNormPlus and NormFinder, PGK1 and PPIA are more stable glomerular reference transcripts compared to GUSB, GAPDH, POLR2A, RPLPO, TBP, B2M, ACTB, 18SrRNA and HMBS.ConclusionsOur approach implements compartment-specific glomerular mRNA expression analysis into research applications, even regarding glomerular substructures like parietal epithelial cells. We recommend using of at least 60 micro-dissected unstained glomerular or 300 hemalaun-stained Bowman’s capsule transections to obtain sufficient input mRNA for reproducible results. Hereby, the range of RNA concentrations in 60 micro-dissected glomeruli is low and appropriate normalization of Cq values using our suggested reference transcripts (PGK1 and PPIA) allows compensation with respect to different amounts of RNA purity and quantity.

Extraction of high-quality RNA from Bacillus subtilis with a lysozyme pre-treatment followed by the Trizol method

A suitable technique (lysozyme combined with Trizol reagent) was developed to improve the RNA yield, purity and integrity from Bacillus subtilis, under different bacterial growth stages. The obtained RNA was intact, having the required characteristics for downstream applications.

Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays

AimsHistopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS)...

Evaluation of RNA from human trabecular bone and identification of stable reference genes.

The isolation of good quality RNA from tissues is an essential prerequisite for gene expression analysis to study pathophysiological processes. This study evaluated the RNA isolated from human trabecular bone and defined a set of stable reference genes. After pulverization, RNA was extracted with a phenol/chloroform method and then purified using silica columns. The A260/280 ratio, A260/230 ratio, RIN, and ribosomal ratio were measured to evaluate RNA quality and integrity. Moreover, the expression of six candidates was analyzed by qPCR and different algorithms were applied to assess reference gene stability. A good purity and quality of RNA was achieved according to A260/280 and A260/230 ratios, and RIN values. TBP, YWHAZ, and PGK1 were the most stable reference genes that should be used for gene expression analysis. In summary, the method proposed is suitable for gene expression evaluation in human bone and a set of reliable reference genes has been identified.

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.

Optimization of phenol-chloroform RNA extraction

Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis.•Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.

Comparison of Different Methods of RNA Preparation from Peripheral Blood for Nucleic Acid Amplification Assay

Background: Nucleic acid amplification assays (NAAs), such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are used for disease diagnosis. Current nucleic acid isolation kits require several hours for completion of protocol including the complicated handling steps. Objective: In this study, a simple and cost-effective nucleic acid preparation method was developed, and its performance was compared with those of commercial kits. Materials and Methods: RNA was prepared using our method and three commercial RNA isolation kits. The RNA quantity and quality were evaluated using the NanoDrop spectrophotometer and Agilent 2100 bioanalyser. Reverse transcription LAMP (RT-LAMP) reactions were performed to determine the usability of the RNA preparation methods. Results: The concentrations of RNA extracted from blood samples by four different methods were sufficient for use in NAAs. The RNA integrity number was >7.0 when RNA was isolated using other RNA isolation kits but lower when prepared using our method. The RT-LAMP reaction was successfully performed when RNA was prepared using any of the methods. Conclusions: These results demonstrate that despite the lower purity and integrity of RNA, our RNA preparation protocol is simple and rapid and shows reasonable performance in RT-LAMP.

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.

Feasibility of the salivary transcriptome as a novel biomarker in determining disease susceptibility.

The salivary transcriptome may present as a readily available and non-invasive source of potential biomarkers. The development of chronic periodontitis is determined by individual patient susceptibility; hence, the aim of this study was to determine the potential of the salivary transcriptome as a biomarker of disease susceptibility using chronic periodontitis as an example. Using an Oragene® RNA kit, the total RNA was purified from the saliva of 10 patients with chronic periodontitis and 10 patients without chronic periodontitis. The quantity and quality of the total RNA was determined, and a measure of gene expression via cDNA was undertaken using the Affymetrix microarray system. The microarray profiling result was further validated by real-time quantitative polymerase chain reaction. Spectrophotometric analysis showed the total RNA purified from each participant ranged from 0.92μg/500μL to 62.85μg/500μL. There was great variability in the quantity of total RNA obtained from the 2 groups in the study with a mean of 10.21±12.71μg/500μL for the periodontitis group and 15.97±23.47μg/500μL for the control group. Further the RNA purity (based on the A260 /A280 ratio) for the majority of participants (9 periodontitis and 6 controls) were within the acceptable limits for downstream analysis (2.0±0.1). The study samples, showed 2 distinct bands at 23S (3800bp) and 16S (1500bp) characteristic of bacterial rRNA. Preliminary microarray analysis was performed for 4 samples (P2, P6, H5 and H9). The percentage of genes present in each of the 4 samples was not consistent with about 1.8%-18.7% of genes being detected. Quantitative real-time polymerase chain reaction confirmed that the total RNA purified from each sample was mainly bacterial RNA (Uni 16S) with minimal human mRNA. This study showed that minimal amounts of human RNA were able to be isolated from the saliva of patients with periodontitis as well as controls. Further work is required to enhance the extraction process of human mRNA from saliva if the salivary transcriptome is to be used in determining individual patient susceptibility.

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.

Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum

Exosomes are cell-derived vesicles and are abundant in biological fluids; they contain RNA molecules which may serve as potential diagnostic biomarkers in 'precision medicine'. To promote the clinical application of exosomal RNA (exoRNA), many isolation methods must be compared and validated. Exosomes in cell culture medium (CCM) and serum may be isolated using ultracentrifugation (UC), ExoQuick or Total Exosome Isolation Reagent (TEI), and exoRNA may be extracted using TRIzol-LS, SeraMir, Total Exosome RNA Isolation (TER), HiPure Liquid RNA/miRNA kit (HLR), miRNeasy or exoRNeasy. ExoRNA was assessed using NanoDrop, Bioanalyzer 2100, quantitative polymerase chain reaction and high-throughput sequencing. UC showed the lowest recovery of particles, but the highest protein purity for exosome isolation. For isolation of exoRNA, we found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM. High yield and a narrow size distribution pattern of small RNA were shown in exoRNA isolated by exoRNeasy from serum. In RNA profile analysis, the small RNA constituent ratio, miRNA content and amount varied as a result of methodological differences. This study showed that different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA. Herein we discuss the advantages and disadvantages of each method and their application to different materials, therefore providing a reference according to research design.

Analytic and Diagnostic Performances of Human Papillomavirus E6/E7 mRNA Test on up-to 11-Year-Old Liquid-Based Cervical Samples. A Biobank-Based Longitudinal Study.

Human Papillomavirus (HPV) E6/E7 mRNA test demonstrated high specificity in detecting HPV infections, but studies assessing its efficacy in terms of cancer risk stratification are lacking. Follow-up studies are arduous and expensive. Biobank would be the answer to the problem, although data investigating the effects of long-term storage on RNA preservation are still needed. We addressed these issues by retrieving 202 residual liquid-based cervical specimens, collected from 149 women attending cervical cancer screening during the years 2001–2012. Samples were stored in Adriatic Biobank at room temperature and without any handing. After calculation of RNA yield and purity, E6/E7 mRNA test was retrospectively performed on each samples, to assess analytic and diagnostic performances. Using automated extraction procedures, RNA of good quantity and quality was obtained. The mean value of RNA concentration was 27.5 ng/μL. The mean A260/A280 ratio was 2.1. An invalid mRNA test result was found in 11.9% of the specimens. Neither RNA integrity, nor analytic performances of mRNA test were influenced by the year of sample collection. In total, 62.4% of the specimens tested as mRNA positive; among these, 89.2% were CIN2+. E6/E7 mRNA was detected in all Squamous Cervical Cancer (SCC) cases. Percentage of positive samples increased with the severity of histological diagnosis. mRNA testing, showing specificity and predictive values of 75.6% and 84.4%, respectively, significantly improved the corresponding values for DNA testing. Thus, the reflex mRNA test was demonstrated to be suitable to triage women with persistent cervical lesions. A “one sample for all” approach is possible, with practical benefits for Biobank-based long-term longitudinal studies, diseases prevention, prediction, diagnosis and treatment.

Prostaglandin synthesis enzymes' gene transcription in bitches with endometritis.

Endometritis is a major cause of infertility in many domestic species. However, until now the pathogenesis of the endometritis in the bitch is unclear. The aim of this study was to evaluate the gene transcription pattern of prostaglandin (PG) synthesis enzymes (cyclooxygenase [COX2], PTGES-1 and PGFS) in the endometrium of bitches with or without endometritis. Thirty mixed breed bitches in dioestrus, aged between 1 and 5years, and weighing between 10 and 30kg were used. After ovariohysterectomy (OVX), uterine biopsy samples were collected from the middle part of both horns. Then, endometrial epithelium was collected using the cytobrush method and mRNA analysis was performed by real-time RT-PCR. Data were analysed with Kruskal-Wallis anova using the sas® software. Uterine condition was identified by endometrial biopsies (normal endometria [n=11; NE], acute endometritis [n=10; AE] and chronic endometritis [n=9; CE]). The COX2, PTGES-1 and PGFS/AKR1C3 mRNA expression in bitches with and without endometritis was similar. Except for PGFS/AKR1C3, gene transcription of COX2 and PTGES-1 was significantly increased in AE compared with CE. In addition, COX2 gene transcription was significantly increased in AE compared with NE. In contrast, no differences were found for COX2, PTGES-1 and PGFS/AKR1C3 mRNA expression in the samples of NE compared with CE.

High-yield extraction of Escherichia coli RNA from human whole blood.

Studies of bacterial transcriptomics during bloodstream infections are limited to-date because unbiased extraction of bacterial mRNA from whole blood in sufficient quantity and quality has proved challenging. Problems include the high excess of human cells, the presence of PCR inhibitors and the short intrinsic half-life of bacterial mRNA. This study aims to provide a framework for the choice of the most suitable sample preparation method. Escherichia coli cells were spiked into human whole blood and the bacterial gene expression was stabilized with RNAprotect either immediately or after lysis of the red blood cells with Triton X-100, saponin, ammonium chloride or the commercial MolYsis buffer CM. RNA yield, purity and integrity were assessed by absorbance measurements at 260 and 280 nm, real-time PCR and capillary electrophoresis. For low cell numbers, the best mRNA yields were obtained by adding the commercial RNAprotect reagent directly to the sample without prior lyses of the human blood cells. Using this protocol, significant amounts of human RNA were co-purified, however, this had a beneficial impact on the yields of bacterial mRNA. Among the tested lysis agents, Triton X-100 was the most effective and reduced the human RNA background by three to four orders of magnitude. For most applications, lysis of the human blood cells is not required. However, co-purified human RNA may interfere with some downstream processes such as RNA sequencing. In this case, blood cell lysis with Triton X-100 is desirable.

Optimization of high quality total RNA isolation from the microalga, Chlorella sp. (Trebouxiophyceae, Chlorophyta) for next‐generation sequencing

SUMMARYThe advent of high‐throughput next‐generation sequencing (NGS) has enabled more comprehensive transcriptome analyses to obtain gene expression data and understand metabolic pathways essential for functional analysis and systems biology. The isolation of high purity and intact RNA of sufficient quality and yield is crucial to the success of NGS sequencing. In green microalgae, extraction of nucleic acids is hindered by high concentration of lipids and polysaccharides that co‐precipitate with nucleic acids thus resulting in reduced yield and poor quality extracts. The present study compared the performance of standard and modified total RNA isolation protocols using different cell disruption techniques combined with TRIzol reagent or a commercially available kit, in meeting the stringent requirements for downstream NGS application of a green freshwater microalga, Chlorella sp. The protocols were evaluated for (i) the yield of RNA, (ii) integrity of RNA as determined by the RNA integrity number and (iii) the purity of RNA as determined by the A260/A280 and A260/A230 ratios. In general, higher yields were obtained by cell disruption via flash freezing and homogenizing with liquid nitrogen followed by lysis with TRIzol reagent. We recommend the incorporation of a salt precipitation step to improve the purity and integrity of RNA isolated. Results of this study served as a simple and low‐cost practical guide for researchers working on isolation of high quality total RNA from microalgae, considering that most relevant publications do not provide a detailed methodology for total RNA isolation or use more expensive methods (e.g. bead‐beater).