Comparison of Automated and Manual Viral Nucleic Acid Extraction Kits for Covid-19 Detection Using qRT-PCR

@#Introduction: The emergence of a novel Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a pandemic. Rapid and accurate diagnosis method is crucial to reduce the disease burden and to improve early diagnosis approaches to control of the disease. Real time Reverse transcriptase PCR (qRT-PCR) has been identified by the World Health Organization as the most sensitive and specific method of detection. However, the success of this assay relies on the quantity and quality of the extracted viral RNA. Methods: Various methods have been developed for nucleic acid extraction however, the methods have not been assessed. RNA extraction was performed from 24 nasopharyngeal swab samples using a manual extraction kit (GF-1) and an automated extraction kit (Genolution). The concentration and purity of the extracted RNA samples were measured, and its performance were tested using qRT-PCR. Results: The average concentration and purity of the RNA samples extracted using GF-1 kit was higher compared to Genolution. Similarly, the qRT-PCR assay using the RNA samples extracted using manual extraction was better compared to automated kit. Conclusion: Both the manual and automated extraction kits have its advantages and disadvantages in terms of yield and purity. However, with proper optimization, both methods may be used for routine molecular diagnostic of COVID-19 in laboratories.

An easy and robust method for the isolation of high quality RNA from coconut tissues

BACKGROUND: Coconut tissues consist of a complex network of polysaccharides, proteins, polyphenols, and lipids that can bind to nucleic acids and pose difficulty in isolation. Certainly, a vigorous method is required to isolate high quality and quantity of RNA from such tissues for the purpose of downstream experiments. In this paper, we discuss a newly developed method for the Isolation of RNA from Complex Matrices (IRCM) method from coconut tissues. RESULTS: The method is robust, cheap, and efficient for the extraction of quality RNA in high quantities from the solid endosperm of stored and fresh coconut (150 µg/g FW with A260/280 = 1.89 and 247.5 µg/g FW with A260/280 = 1.91), coconut apple (263.8 µg/g FW with A260/280 = 1.97), and coconut bud (1052.5 µg/g FW with A260/280 = 2.00). The other well established methods, such as Method of RNA Isolation from Palm (MRIP), Cetyl Trimethyl Ammonium Bromide (CTAB), TRIZOL, and RNA plant kit failed to isolate quality RNA in appreciable quantities from the coconut tissues. Furthermore, the resultant RNA performed well in the downstream experiment, that is, RT-PCR for the production and amplification of cDNA. CONCLUSIONS: From the study, we concluded that the present method will play a vital role in the extraction of high quality RNA from complex matrices in a short time.

Modified Trizol Method for Extracting RNA from Mouse Skin / 中山大学学报(医学科学版)

@#【Objective】Due to the tough nature of skin tissue and a high presence of RNases，the isolation of skin RNA by the classical Trizol method presents a challenge. Therefore，we adapted and tested different sample treatment protocols to improve the Trizol method for high- quality extraction of skin RNA.【Methods】In this study，normal skin of mice processed by different treatments（Tri：submersion Trizol；Pro：RNA sample protector；Cry：cryopreservation in liquid nitrogen frozen and then - 80 ℃ refrigerator；LNG：liquid nitrogen grinding；Cut：scissor cutting）were used as the experimental groups. Spinal cord tissue（Sp）was used as the reference group，and skin tissue of mouse psoriasis model induced by imiquimod（IMQ）was used as the validation group. We compared skin RNA concentration，purity and integrity， as well as IL- 1β mRNA expression extracted by conventional Trizol methods（1-Tri，Nor）and modified Trizol methods（2-Tri，LNG-Tri，Tri-Cut，Pro），which were determined by UV spectrophotometry，agar gel electrophoresis and quantitative reverse transcription PCR（qRT- PCR）.【Results】① Compared with spinal cord（Sp），the total RNA of normal skin tissue extracted with the same classical Trizol method（1-Tri）was with lower yields，more obvious DNA contamination and 5S RNA bands，and higher IL-1β mRNA relative expression，suggesting that skin tissue was relatively special and the classical Trizol methods of skin RNA extraction should be improved. ② Among the different treatment methods of skin tissue，2-Tri and LNG-Tri methods resulted in higher RNA concentrations，lower RNA degradation and lower DNA contamination，and the expression of IL-1β mRNA was closer to normal levels. More importantly，the skin RNA samples extracted by the 2-Tri method can reflect more realistically the variation of IL-1β mRNA expression between normal and psoriasiform groups.【Conclusion】Improved 2-Tri or LNG-Tri method has the advantage of high quality of total RNA，and 2-Tri can more reliably reflect the mRNA expression pattern under physiological and pathological conditions.

False-negative result in molecular diagnosis of SARS-CoV-2 in samples with amplification inhibitors / Resultado falso negativo no diagnóstico molecular de SARS-CoV-2 em amostras com inibidores de amplificação

ABSTRACT Introduction: Although reverse transcription-polymerase chain reaction (rRT-PCR) is the gold standard method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), some factors, such as the presence of amplification inhibitors, lead to false-negative results. Objective: Here we describe the differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to the presence of amplification inhibitors. Material and method: Viral ribonucleic acid (RNA) from samples of nasopharyngeal swabs from 20 patients previously detected as "Negative" and 21 patients detected as "Positive" for SARS-CoV-2 was performed with the EasyExtract DNA-RNA kit (Interprise®). The rRT-PCR was performed with the OneStep/COVID-19 kit (IBMP), with normal and diluted (80 µl of H2O RNAse free) samples, totaling 82 tests. Results: The results indicate that there is an average variation (a < 0.05) delaying the Cq between the results of amplification of the internal control (IC), N gene (NG), and ORF1ab (OF), 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. Discussion: The extraction kit does not completely purify the inhibitor compounds; therefore, no amplified product result may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution; this process reduces the efficiency of rRT-PCR to 29.8% in detecting SARS-CoV-2. Conclusion: Knowing the rRT-PCR standards of diluted samples can assist in the identification of false-negative cases and, consequently, avoid incorrect diagnosis.

RESUMEN Introducción: Aunque la reacción en cadena de la polimerasa con transcriptasa reversa en tiempo real (rRT-PCR) sea el método de referencia para detección del coronavirus tipo 2 del síndrome respiratorio agudo grave (Sars-CoV-2), algunos factores como la presencia de inhibidores de amplificación conducen a resultados falsos negativos. Objetivo: Describimos las diferencias entre los resultados de rRT-PCR para infección por Sars-CoV-2 en muestras normales y diluidas, simulando la necesidad de dilución debido a la presencia de inhibidores de amplificación. Material y método: La extracción de ácido ribonucleico (ARN) viral de muestras de hisopos nasofaríngeos de 20 pacientes previamente detectados como "negativos" y 21 pacientes detectados como "positivos" para Sars-CoV-2 se realizó con el kit Easy Extract DNA-RNA (Interprise®). La rRT-PCR se realizó con el kit OneStep/Covid-19 (IBMP), con muestras normales y diluidas (80 µl de H2O libre de ARNasa), totalizando 82 pruebas. Resultados: Los resultados indican que hay una variación media (a < 0,05) retrasando el ciclo de cuantificación (Cq) entre los resultados de amplificación del control interno (CI), gen N (GN) y ORF1ab (OF) de 1,811 Cq, 3,840 Cq y 3,842 Cq. Discusión: El kit de extracción no purifica completamente los compuestos inhibidores; por lo tanto, puede ocurrir no amplificación. Obtuvimos un diagnóstico falso negativo de 19,04% después de la dilución de la muestra; ese proceso reduce la eficiencia de la rRT-PCR hacia 29,8% en la detección de Sars-CoV-2. Conclusión: Conocer los patrones de la rRT-PCR de muestras diluidas puede ayudar en la identificación de casos falsos negativos y, por consiguiente, evitar un diagnóstico equivocado.

RESUMO Introdução: Embora a reação em cadeia da polimerase de transcrição reversa (rRT-PCR) seja o método padrão-ouro para detecção de coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2), alguns fatores como a presença de inibidores de amplificação levam a resultados falso negativos. Objetivo: Descrevemos as diferenças entre os resultados de rRT-PCR para infecção por SARS-CoV-2 em amostras normais e diluídas, simulando a necessidade de diluição devido à presença de inibidores de amplificação. Material e método: A extração de ácido ribonucleico (RNA) viral de amostras de suabes nasofaríngeos de 20 pacientes previamente detectados como "negativos" e 21 pacientes detectados como "positivos" para SARS-CoV-2 foi realizada com kit o EasyExtract DNA-RNA (Interprise®). A rRT-PCR foi realizada com o kit OneStep/COVID-19 (IBMP), com amostras normais e diluídas (80 µl de H2O RNAse-free), totalizando 82 testes. Resultados: Os resultados indicam que existe uma variação média (a < 0,05) atrasando o Cq entre os resultados de amplificação do controle interno (CI), gene N (GN) e ORF1ab (OF) de 1,811 Cq, 3,840 Cq e 3,842 Cq, respectivamente. Discussão: O kit de extração não purifica completamente os compostos inibidores, portanto, pode ocorrer não amplificação. Obtivemos um diagnóstico falso negativo de 19,04% após a diluição da amostra; esse processo reduz a eficiência da rRT-PCR para 29,8% na detecção de SARS-CoV-2. Conclusão: Conhecer os padrões da rRT-PCR de amostras diluídas pode auxiliar na identificação de casos falso negativos e, consequentemente, evitar um diagnóstico incorreto.

Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies / Extração e purificação de RNA proveniente de dentina humana cariada: uma abordagem para viabilizar estudos de expressão gênica

Background: RNA isolation from bacteria within dentine caries lesions could be difficult due to reduced amount of collectable biomass and high mRNA instability. Attempting to overcome this challenge we describe one protocol developed to extract and purify total RNA from dentine lesions. Objective: customize a bacterial RNA extraction and purification method from human carious dentine. Methods: quantity and purity of extracted RNA were measured with a microvolume UV-VIS spectrophotometer, RNA integrity was assessed by standard denaturing agarose gel electrophoresis and images were captured under ultraviolet light with camera and analyzed. DNase treatment removed genomic DNA and an additional step of purification was carried out in silica spin column. Results: final yield (ng/µl) was 67.01 ± 22.33, absorbance ratio A260/A280 = 2.0 ± 0.07 and RNA integrity were obtained. The purified samples were reversely transcribed and the expression of atpD and fabM gene from Streptococcus mutans analyzed by quantitative real-time PCR. Conclusion: the extraction methodology developed produced high-quality RNA from dentine microbiota for transcriptional analysis.

Introdução: o isolamento de RNA de bactérias dentro de lesões de dentina cariada pode ser difícil devido à quantidade reduzida de biomassa e alta instabilidade de mRNA. Na tentativa de superar esse desafio, descrevemos um protocolo desenvolvido para extrair e purificar o RNA total das lesões dentinárias. Objetivo: personalizar um método de extração e purificação de RNA bacteriano a partir da dentina cariada humana. Métodos: a quantidade e a pureza do RNA extraído foram medidas com um espectrofotômetro UV-VIS de microvolume, a integridade do RNA foi avaliada por eletroforese em gel de agarose desnaturante padrão e as imagens foram capturadas sob luz ultravioleta e analisadas. O tratamento com DNase removeu o DNA genômico e uma etapa adicional de purificação foi realizada em coluna de spin de sílica. Resultados: o rendimento final (ng / µl) foi de 67,01 ± 22,33, a razão de absorbância A260 / A280 = 2,0 ± 0,07 e a integridade do RNA foram obtidas. As amostras purificadas foram transcritas reversamente e a expressão do gene atpD e fabM de Streptococcus mutans analisadas por PCR quantitativo em tempo real (RT-qPCR). Conclusão: a metodologia de extração desenvolvida produziu RNA de alta qualidade da microbiota dentinária para análises transcricionais.

Extraction of high-qualityrna from tumours tissues using mechanical homogenisation method for downstream application / 国际药学研究杂志

RNA quality and quantity are crucial factors in ensuring the accuracy of RNA-based downstream applications such as the northern blotting, qPCR, transcriptome and microarray analyses. Bead beating is an effective mechanical lysis method that candisrupt a wide range of biological samples including microorganisms, plants, animals and human tissues. In this experiment, a MINI bead beater, TRIzol™ reagent and zirconium beads were used to demonstrate the bead-beating method in extracting high-quality total RNA from mouse tumour tissues from four different anatomies. The tumour tissues were isolated from the Cdkn2a-/-, Tyr-HRASG12Vmouse model of melanoma that develops spontaneous melanomas. The extracted total RNA was measured through absorbance reading to verify the purity and yield of the product. Whilst, the intactness of the final product was examined using ethidium bromide stained agarose gel electrophoresis. To obtain an overall reflection of the RNA quality, extracted RNAs were subjected to qRT-PCR to evaluate endogenous mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH)gene expressions. The results of the experiment further affirmed that the application of bead beater along with TRIzol™ reagent and zirconium beads has the potential of extracting high-quality RNA from tumour tissues without affecting the yield, purity, integrity and functionality of the RNA.

Molecular cloning and characterisation of scavenger receptor class B in pearl oyster Pinctada fuctada martensii

Background: Molluscs can accumulate carotenoids in their body tissues by predominantly feeding on aquatic plant sources. Carotenoid transport and absorption are determined by the regulation of various proteins such as Scavenger receptor class B(SR-BI). We report the identification and characterisation of pearl oyster Pinctada fuctada martensii SR-BI (PmSR-BI). The correlation between total carotenoid content (TCC) and gene expression was also estimated. Results: The full-length cDNA of PmSR-BI was 1828 bp, including an open-reading frame encoding of 1518 bp with a pI value of 5.83. PmSR-BI protein contains a hydrophobic CD36 domain and four centrally clustered cysteine residues for the arrangement of disulphide bridges. The deduced amino acid sequence had an identity of 30% to 60% with the SR-B of other organisms. Reverse transcription polymerase chain reaction analysis showed that mRNA transcripts were expressed in multiple tissues of adult pearl oyster. A higher expression of PmSR-BI gene was observed in the hepatopancreas than in the adductor muscle, gill and mantle. The TCC and gene expression of PmSR-BI were significantly correlated (P b 0.05), with a correlation coefficient of 0.978. Conclusions: The results suggested that PmSR-BI is involved in the absorption of carotenoids in the pearl oyster P. fuctada martensii.

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

Background: Small ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum (Prunus domestica) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV-Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry. Results: We describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV­P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159. Conclusion: We propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species.

Expressão gênica de IGF-I e GHR no fígado e no músculo do peito de codornas de corte suplementadas com diferentes níveis de metionina em duas gerações sucessivas / IGF-I and GHR gene expression in liver and breast muscle of meat quails supplemented with different levels of methionine in two successive generations

Este estudo foi desenvolvido com o objetivo de avaliar a expressão gênica do fator de crescimento semelhante à insulina I (IGF-I) e do receptor do hormônio do crescimento (GHR) no fígado e no músculo do peito de codornas de corte, alimentadas com dietas contendo diferentes níveis de suplementação de metionina, em duas gerações sucessivas. Foram utilizadas codornas dos 22 aos 42 dias de idade, distribuídas em três e cinco tratamentos na primeira e na segunda geração, respectivamente. Ao final, as aves foram abatidas por deslocamento cervical, sendo coletados fígado e músculo do peito para extração de RNA total. O cDNA foi amplificado usando primers específicos para os genes analisados. Os resultados mostraram que a expressão dos genes GHR e IGF-I sofreu influência da suplementação. No quinto tratamento, em que apenas a primeira geração recebeu uma suplementação acima do padrão das exigências para o período, houve uma expressão significativamente maior do GHR tanto no músculo do peito como no fígado e igualmente do IGF-I no músculo, levando a concluir que o excesso de metionina na dieta torna-se tóxica para as aves. Apesar de a expressão dos genes ter sofrido influência da adição de metionina nos níveis estudados, não foi observada diferença no consumo alimentar, na conversão alimentar e no peso das aves.(AU)

This study was conducted to evaluate the gene expression of the insulin-like I growth factor (IGF-I) and growth hormone receptor (GHR), in the liver and chest muscle of slaughter quails fed with diets containing different levels of methionine supplementation, in two successive generations. Twenty-two to 42 day-old quails were used, distributed in three and five treatments in the first and second generation, respectively. At the end, the birds were killed by cervical dislocation, and their liver and chest muscle were collected for total RNA extraction. The cDNA was amplified using specific primers for the genes analyzed. The results showed that the expression of GHR gene and IGF-I were influenced by the supplementation. In the fifth treatment, where only the first generation received supplementation above the standard requirements for the period, there was a significantly higher expression of GHR both in muscle chest and in the liver, and also IGF-I on muscle, leading to the conclusion that the excess dietary methionine becomes toxic to birds. Despite the gene´s expression seeming to be influenced by the addition of methionine levels in the study, there was no difference in feed intake, feed conversion and weight of the birds.(AU)

Comparison of three different methods for isolating RNA from Oncomela-nia hupensis / 中国血吸虫病防治杂志

Objective To compare the effects of three kinds of Oncomelania hupensis RNA extraction methods,namely a modified SDS method,TRIzol reagent method,and CTAB method,so as to obtain an economical and efficient method for RNA extraction from O. hupensis. Methods The modified SDS method,TRIzol reagent method and CTAB method were applied to ex-tract the RNA from O. hupensis. A nucleic acid protein analyzer was used to measure the concentration and purity of RNA. The yields were calculated by the concentration of the products. The purity was indicated by A260/A280 and A260/A230. The quality of RNA was inspected by 1% agarose gel electrophoresis. The β-acting gene was selected as the target gene for RT-PCR analysis. Re-sults The RNA yields obtained by using the three kinds of extraction methods were significantly different(F = 16895.85,P <0.01)according to the analysis of variance. The LSD test showed that the yields obtained by using the modified SDS method were the highest,and those obtained by the CTAB method were the lowest. The purity of RNA extracted by the CTAB method was su-perior to that by the other two methods,and the A260/A280 and A260/A230 ratios of the CTAB method were in the range from 1.8-2.0 and 2.0-2.2. The A260/A230 ratios of the other two methods were both lower than 2.0. The RNA extracted by the modified SDS meth-od had the better integrity. The electrophoresis results showed that the 28S rRNA band,18S rRNA band and 5S rRNA band were clear,and there was no obvious smear between each band. The RNA obtained by the TRIzol reagent method had no 28S rRNA band,and that obtained by the CTAB method had no 28S rRNA and 5S rRNA bands. The β-acting gene of the RNA ex-tracted by all the three methods could be amplified by RT-PCR. The costs and time-consuming of the modified SDS method were less than those of the other two methods. Conclusion The modified SDS method is an economic and efficient method,and it is suitable for extracting the RNA of O. hupensis,especially for large sample preparation.

Feasibility of Detection Three Kinds of Sexually Transmitted Pathogenby MGX Automatic Nucleic Acid Extraction Workstation / 现代检验医学杂志

Objective To verify the feasibility of Chlamydia trachomatis(CT),Neisseria gonorrhoeae(NG),Ureaplasma urealyticum(UU)detection by automatic nucleic acid extraction workstation(MagX).Methods The test samples of CT (159),NG(128)and UU(144)were collected.The samples were extracted of nucleic acid two methods:manual extraction, which entailed manually preparing PCR reaction system and MagX automatic nucleic acid extraction workstation,which au-tomatically prepared the reaction system.The two parts of nucleic acids proceeded to the Simultaneous Amplification and Testing(SAT).The result of manual extraction was set as the golden standard and the Kappa consistency analysis was con-ducted.Meanwhile,the sensitivity,specificity,accuracy and carry pollution experiments of MagX were verified.Results The sensitivity of MagX automatic nucleic acids extraction workstation were 92.59%(CT),100.00%(NG)and 93.33%(UU).The specificity were 98.48%(CT),98.18%(NG)and 95.24%(UU).The accuracy were 97.48%(CT),98.44%(NG)and 94.44%(UU).The results of kappa consistency analysis were greater than 0.8(CT:Kappa=0.910 6,NG:Kap-pa=0.938 3,UU:Kappa=0.885 6).MagX detected the precision of CT,NG and UU:Coefficient of Variation(CV)<5%. There was no pollution phenomenon.Conclusion MagX automatic nucleic acids extraction workstation could be used to test three kinds of sexually transmitted pathogen in clinical settings.

An Improved Total RNA Extraction Method for White Jelly Mushroom Tremella fuciformis Rich in Polysaccharides

An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus—Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and 213 µg/g fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides.

Performance evaluation of the one self-developed device for transmitting negative pressure in RNA extraction / 国际检验医学杂志

Objective To evaluate the self‐developed device for transmitting negative pressure with large capacity before propa‐gating .Methods Totally 67 ribonucleic acid(RNA) of WBC > 2 .0 × 109 /L fresh whole blood were extracted with the intrinsic de‐vice(control) and the self‐developed device for transmitting negative pressure with large capacity(test) in parallel .The difference of the performance including efficiency ,concentration ,purity and integrity of RNA extraction were evaluated between the two groups . Results In 67 specimens of RNA extraction ,efficiency ,concentration ,purity and integrity of the control device were 0 .97(portion/minute) ,(248 .8 ± 21 .4)μg/mL ,(1 .995 ± 0 .095) (OD260/OD280) ,(2 .020 ± 0 .082) (OD260/OD230) ,1 .964 - 2 .025 (95% con‐fidence interval for mean) (OD260/OD280) ,2 .001 - 2 .040 (95% confidence interval for mean) (OD260/OD230) and complete . Those of test device were 1 .63 portion/minutes ,(260 .3 ± 21 .8)μg/mL ,(2 .093 ± 0 .092) at OD260/OD280 ,(2 .071 ± 0 .120) at OD260/OD230 ,2 .075 - 2 .113 for 95% confidence interval for mean at OD260/OD280 ,2 .044 - 2 .103 for 95% confidence interval for mean at OD260/OD230 and complete .The t value was 24 .570 (P< 0 .001) in paired t‐test ,and the linear regression equation was Y = 0 .950X + 0 .039 ,R2 = 0 .903 for the two groups data of RNA concentration .Conclusion The self‐developed device for transmitting negative pressure with large capacity is excellent in the work efficiency ,concentration ,purity ,integrality and of the RNA in extracting .It is better than the intrinsic device in the clinical value for situation of lab .

An Effective Method of RNA Extraction from Mycobacterium tuberculosis / 대한임상미생물학회지

In the RNA-based study, it is important to extract high-quality RNA. However, RNA extraction from Mycobacterium tuberculosis is problematic due to its thick, waxy cell wall rich in mycolic acid, which renders the cells resistant to lysis. Using TRIzol reagent and several powerful bead-beating steps, a high quantity of RNA was obtained.

A Melting Method for RNA Extraction from the Mucosal Membrane of the Mouse Middle Ear

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.

A Melting Method for RNA Extraction from the Mucosal Membrane of the Mouse Middle Ear

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.

Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani

Background Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is one of the most important pathogens of rice (Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani, five methods, including E.Z.N.A.™ Fungal RNA Kit, sodium dodecyl sulfate (SDS)-sodium borate, SDS-polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study. Results The electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS-sodium borate, SDS-PVP and E.Z.N.A.™ Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment. Conclusion It is concluded that SDS-sodium borate and SDS-PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A.™ Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield.

Improvement in RNA extraction from S. cerevisie by optimization in the autolysis and NH3 hydrolysis

The optimization of autolysis of Saccharomyces cerevisiae from brewery was studied aiming at the maximum ribonucleic acid extraction and yeast extract production. The best conditions for yeast autolysis was 55.2ºC, pH= 5.1 and 9.8 percent NaCl for 24h of processing, without the NH3 use. In these conditions, the RNA yield was 89.7 percent, resulting in 51.3 percent of dehydrated yeast extract with 57.9 percent protein. The use of 12.2 percent NH3 at 60ºC after autolysis (8h) and plasmolysis (8h) was not viable due to the reduction in the RNA yield from 89.7to78.4 percent. On the other hand, the thermal shock at 60ºC for 15 minutes prior to autolysis provided an increase in the yield from 89.7 to 91.4 percent. The autolysis, including NaCl plasmolysis in the optimized conditions was efficient, economic and with short time, thus usable for industrial purpose to obtain more valuable products such as yeast extract enriched in RNA and/or protein, for different applications.

Detection of Rare Cancer Cells in the Blood by RNA Extraction of Filtered Mononuclear Cells and Reverse Transcription-PCR / 임상검사와정도관리

BACKGROUND: To enhance the cancer cell detection rate in blood, we tried to detect cancer cells by blood filtration, RNA extraction and reverse transcription (RT)-PCR in the filtered mononuclear cells (MNCs). METHODS: From the specimens of whole blood and filtered MNCs, RNA was extracted by the guanidinium isothiocyanate buffer method. Filtration efficiency was evaluated by measurement of leukocyte count, red cell count, and hemoglobin level. To compare the RNA extraction efficiency between whole blood and filtered MNCs, the followings were examined: (1) RNA electrophoresis, (2) hTERT, survivin, plakophilin, LunX, and MAGE A1-6 RT-PCR, and (3) the detection limit of added SNU484 cells in the blood by MAGE A1-6 RT-PCR. Finally blood specimens of 13 lung cancer patients were used to detect cancer cells by LunX and MAGE A1-6 RT-PCR with filtered MNCs. RESULTS: The filtration method revealed 0%, 92.8% and 95.1% filtration rates for leukocyte, red cell, and hemoglobin, respectively. Contamination of concentrated genomic DNA was observed in the electrophoresis of RNA extracted from whole blood. Positive rates of hTERT, survivin and plakophilin were higher in the filtered MNCs. The filtration method detected 1 SNU484 cell/mL, and the blood samples of 4 (30.8%) lung cancer patients showed positive results for RT-PCR. CONCLUSIONS: For the detection of cancer cells in the blood, the filtration method was very efficient, and LunX and MAGE A1-6 genes would be useful for the detection of blood cancer cells in patients with lung cancer.

Isolation of total RNA from hard bamboo tissue rich in polyphenols and polysaccharides for gene expression studies

RNA isolation from hard and woody internodal bamboo (Bambusa balcooa) tissue is very difficult due to the presence of secondary metabolites, polysaccharides, and polyphenolics. These compounds often co-precipitate with isolated RNA and hinder downstream applications. We have developed an efficient, cost effective and reproducible RNA isolation method from hard tissue of bamboo internode. This protocol includes an additional organic solvent refinement steps to remove endogenous phenolic compounds and acidic phenol (pH 4.2) to critically stabilize RNA in extraction buffer. In addition to these, two 2M Lithium chloride washing steps were introduced to eliminate DNA and polysaccharides contamination. The RNA isolated from the present protocol was found to be superior, when compared to total RNA extracted by other available protocols. The A260/A280 absorption ratio of the isolated RNA was found ranging between 1.89-1.97. The integrity of 28S and 18S rRNA was highly satisfactory when analyzed in agarose denaturing gel. RNA was further used for RT PCR, northern hybridization, cDNA library and subtractive hybridization without any further refinement.