A Simple and Easy Method for RNA Extraction from the Cyanobacterium Synechocystis sp. PCC 6803.

Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC)

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.

Ribonucleic acid (RNA) extraction is the necessary first step in many protocols, primarily to investigate genes and gene expression. RNA comes in a variety of forms: total RNA, ribosomal RNA, messenger RNA (mRNA), and small interfering RNA (siRNA) to name a few. In some instances, total RNA is all that is required; however most applications will require the enrichment for some particular form of RNA. In plants, including cereals, total RNA is a mixture of many types of RNA and enrichment is generally required. In this protocol, the TRIzol(®) method of RNA extraction from cereal leaf material is described, as it is a relatively simple technique.

Background: Next generation sequencing (NGS) and especially ribonucleic acid (RNA) sequencing is a powerful tool to acquire insights into molecular disease mechanisms. Therefore, it is of interest to optimize methods for RNA extraction from archival, formalin fixed and paraffin embedded (FFPE) tissues. This is challenging due to RNA degradation and chemical modifications. The aim of this study was to find the most appropriate method to extract RNA from FFPE renal tissue to enable NGS.Method: We evaluated seven commercially available RNA extraction kits: High Pure FFPE RNA Isolation (Roche), ExpressArt Clear FFPE RNAready (Amsbio), miRNeasy FFPE, RNeasy FFPE (Qiagen), PureLink FFPE Total RNA (Invitrogen), RecoverAll Total Nucleic Acid Isolation (Ambion) and Absolutely RNA FFPE Kit (Agilent). RNA was obtained from tissue blocks of two healthy, male Wistar rats and from normal renal tissue of patients undergoing nephrectomy. Yield and quality of RNA extracted from rat whole kidney sections, human kidney core biopsies and laser capture microdissected (LCM) glomerular cross-sections were assessed: Analyses of RNA quantity were performed using NanoDrop and Qubit. RNA quality is reflected by DV200 values (% of RNA fragments >200 nucleotides) utilizing the Agilent 2100 BioAnalyzer. RNA of human LCM samples was subsequently sequenced using the Illumina TruSeq® RNA Access Library Preparation Kit.Conclusion: Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.

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.

The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans Moina micrura using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from M. micrura, with the quantity (RNA concentration = 26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15, A280:230 = 1.85 ± 0.09) and integrity (RNA integrity number, RIN = 7.20 ± 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33–35 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.

The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans Moina micrura using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from M. micrura, with the quantity (RNA concentration = 26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15, A280:230 = 1.85 ± 0.09) and integrity (RNA integrity number, RIN = 7.20 ± 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33-35 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.

Ribonucleic acid (RNA) extraction requires meticulous sample handling to ensure purity and integrity. Although a variety of commercial kits are available, along with optimised protocols, pre-extraction sample processing remains a challenging procedure, especially with tissue samples. In our brief report, we describe the beneficial impact of freezing tissues before homogenisation on the quality of RNA extraction. Lung tissues were freshly excised from mice and homogenised with or without prior quick freezing in a freezer. Then, RNA was extracted according to the protocol provided with a commercial column-based RNA extraction kit. RNA quality was analysed by UV absorbance and agarose gel electrophoresis. We found that the frozen tissues yielded better-quality, more intact RNA than the non-frozen tissues, possibly due to lower temperatures during homogenisation. “Smearing”, indicative of RNA degradation, was visible in some of the non-frozen samples. The extra quick-freezing step provides a simple and affordable method for preserving high-quality RNA, especially from tissue samples. Further comparisons can be made to determine whether the observed benefits extend to other tissue types or to quantitative polymerase chain reaction (qPCR) analysis in gene expression studies.

Spleen cells (SpC) from non-immunized rabbits were converted to antibody plaque-forming cells (PFC) by incubation with ribonucleic acid (RNA) extracts of peritoneal exudate cells (PEC) or lymph node cells (LNC) obtained from the immunized rabbits 5 days following one intravenous injection of 4 × 108 sheep red blood cells (SRBC). The RNA extract was inactivated by ribonuclease (RNase) but not by deoxyribonuclease (DNase) or trypsin, indicating that the conversion required intact RNA. Plaque formation was inhibited by 2-mercaptoethanol (2-ME) or goat anti-rabbit γM but not by goat anti-rabbit γG-Fc fragment indicating that the antibody produced by the PFC are of the γM immunoglobulin class. The conversion was specific for the SRBC antigen injected. By use of antibodies to the b4 and b5 allotypic specificities of immunoglobulin light chains, the allotypic specificity of the γM antibody produced by the PFC was identified by direct precipitation of the γM in the plaque or by inhibition of plaque formation. The γM antibody produced by converted SpC invariably possessed the allotypic specificity characteristic for immunoglobulins of the donor of the “immune” RNA extract and not of the donor of SpC. These results could not be explained by contamination of the RNA extracts with anti-SRBC, thus implying that a component of the RNA extract, presumably RNA itself, is providing a code for the synthesis of at least a part of the γM immunoglobulin molecule.

Spleen cells (SpC) from non-immunized rabbits were converted to antibody “indirect” plaque-forming cells (PFC) by incubating them with ribonucleic acid (RNA) extracts of lymph node cells (LNC) obtained from rabbits 18 to 24 days after they were immunized with several intravenous injections of 4 × 108 sheep red blood cells (SRBC). The RNA extract was inactivated by ribonuclease (RNAse) but not by deoxyribonuclease (DNAse) or trypsin, indicating that the conversion required intact RNA. “Indirect” PFC were developed with optimal amounts of goat anti-IgG-Fc fragment and these were not inhibited by 2-mercaptoethanol (2-ME) or goat anti-IgM but were inhibited by excess anti-IgG-Fc fragment. This indicated that the antibodies produced by the PFC are of the IgG immunoglobulin class. The conversion was specific for the SRBC antigen injected. The light chain allotype of the IgG antibody produced by the PFC was identified by use of anti-b4 and anti-b5 to develop the “indirect” PFC, to visualize the PFC by radioautography and to inhibit plaque formation. Most of the “indirect” PFC of the converted SpC (64% to 84%) possessed IgG antibody with the allotype characteristic of the donor of the RNA extract; some of the “indirect” PFC (4% to 32%) possessed IgG antibody with the allotype characteristic of the donor of SpC. When the “RNA-converted” SpC were lysed by freezing and thawing, the anti-SRBC titer of the lysate, measured by the localized hemolysis in gel technique, was 4096 compared to 4 for lysates from the same number of lysed, nonimmune SpC alone. Moreover, anti-SRBC antibody could not be detected in the RNA extracts. These results indicate that a component of the RNA extract, presumably RNA itself, is providing information for the synthesis of at least part of the IgG antibody molecule.

A magnetic material that consists of silica-coated magnetic beads conjugated with graphene oxide (GO) was successfully prepared for facile ribonucleic acid (RNA) extraction. When the GO-modified magnetic beads were applied to separate the RNA from the lysed cell, the cellular RNAs were readily adsorbed to and readily desorbed from the surface of the GO-modified magnetic beads by urea. The amount of RNA extracted by the GO-modified magnetic beads was ≈170 % as much as those of the control extracted by a conventional phenol-based chaotropic solution. These results demonstrate that the facile method of RNA separation by using GO-modified magnetic beads as an adsorbent is an efficient and simple way to purify intact cellular RNAs and/or microRNA from cell lysates.

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.

Ribonucleic acid (RNA) extraction is the first critical step in gene expression analysis. In this chapter, we describe a high-throughput RNA extraction method using guanidine thiocyanate and isopropyl alcohol (HighGI). The use of carboxyl-coated paramagnetic beads, instead of silica membrane columns, enables semi-automation using a liquid handling system and high-throughput RNA extraction for large-scale transcriptome studies. Homemade mixes of paramagnetic beads and buffers make HighGI inexpensive. In addition, HighGI-extracted RNA retains low molecular weight RNA molecules less than 200bp, which is typically lost in commercial column-based kits.

The original single‐step method is the first procedure to isolate purified total ribonucleic acid (RNA) from a variety of sources including tissues and cells from human, animal, plant, yeast, bacterial and viral origins, without the requirement of high‐speed ultracentrifugation. The method is based on liquid‐phase separation resulting in sequestration of pure RNA into the aqueous phase. RNA is precipitated from the aqueous phase, dissolved, reprecipitated and washed with alcohol before the final solubilisation step. The entire procedure can be completed in less than 4 h and it provides RNA that is suitable for many sensitive downstream applications such as RNase protection assays, northern blotting, sequencing studies and reverse transcription‐polymerase chain reaction. This pioneering methodology has served as the impetus for the development of newer and improved RNA extraction methodology that now enable investigators to extract and purify RNA in less than 60 min. Key Concepts: Enzymes within living cells rapidly degrade RNA after a tissue sample is removed from the donor. To prevent RNA degradation, tissue samples that cannot be immediately processed must be rapidly frozen with dry ice or liquid nitrogen and stored frozen at −80 °C until the RNA can be extracted. At the time of RNA extraction, frozen cells or tissues must be immersed in the denaturing solution and rapidly homogenised before the tissue thaws in order to inactivate RNAse and avoid RNA degradation. The quality of the recovered RNA is dependent on a delicate balance of salt concentration and optimal pH. Overloading the extraction solution with too much tissue or diluting the denaturing solution beyond what is specified in the protocol will impact the quality of the resulting RNA. The extraction of RNA from samples that have a high buffering capacity, such as blood, plasma or tissue culture medium, requires greater care in order to maintain optimal salt balance and pH control. Overdrying of the RNA pellets will impede RNA solubilisation. RNA should be solubilised at a concentration that will be appropriate for meaningful spectrophotometric quantitation as well as subsequent downstream molecular biology applications. Enzymes that are involved in RNA degradation are ubiquitous and special care must be taken to avoid RNAse contamination during RNA solubilisation and storage.

The original single‐step method is the first procedure to isolate purified total ribonucleic acid (RNA) from a variety of sources including tissues and cells from human, animal, plant, yeast, bacterial and viral origins, without the requirement of high‐speed ultracentrifugation. This method is based on liquid‐phase separation resulting in sequestration of pure RNA into the aqueous phase. RNA is precipitated from the aqueous phase, dissolved, reprecipitated and washed with alcohol before the final solubilisation step. The entire procedure can be completed in less than 4 h and it provides RNA that is suitable for many sensitive downstream applications such as RNase protection assays, northern blotting, RNA sequencing studies and reverse transcription‐polymerase chain reaction. This pioneering methodology has served as the impetus for the development of newer and improved RNA extraction methodology that now enables investigators to extract and purify RNA in less than 60 min. Some of these newer methods do not require a halogenated organic solvent, or combine the single‐step method with column purification of the RNA. Key Concepts Enzymes within living cells rapidly degrade RNA after a tissue sample is removed from the donor. To prevent RNA degradation, tissue samples that cannot be immediately processed must be rapidly frozen with dry ice or liquid nitrogen and stored frozen at −80°C until the RNA can be extracted. At the time of RNA extraction, frozen cells or tissues must be immersed in the denaturing solution and rapidly homogenised before the tissue thaws in order to inactivate RNAse and avoid RNA degradation. The quality of the recovered RNA is dependent on a delicate balance of salt concentration and optimal pH. Overloading the extraction solution with too much tissue or diluting the denaturing solution beyond what is specified in the protocol will impact the quality of the resulting RNA. The extraction of RNA from samples that have a high buffering capacity, such as blood, plasma or tissue culture medium, requires greater care in order to maintain optimal salt balance and pH control. Overdrying of the RNA pellets will degrade RNA and impede RNA solubilisation. RNA should be solubilised at a concentration that will be appropriate for meaningful spectrophotometric quantitation as well as subsequent downstream molecular biology applications. Enzymes that are involved in RNA degradation are ubiquitous and special care must be taken to avoid RNAse contamination during RNA solubilisation and storage.