Latest News on RNA Isolation: Dec 2020

Latest News on RNA Isolation: Dec 2020

The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on

Since its introduction, the ‘single-step’ method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years. [1]

Importance of RNA isolation methods for analysis of exosomal RNA: Evaluation of different methods

Exosomes are small RNA containing vesicles of endocytic origin, which can take part in cell-to-cell communication partly by the transfer of exosomal RNA between cells. Exosomes are released by many cells and can also be found in several biological fluids including blood plasma and breast milk. Exosomes differ compared to their donor cells not only in size but also in RNA, protein and lipid composition. The aim of the current study was to determine the optimal RNA extraction method for analysis of exosomal RNA, to support future studies determining the biological roles of the exosomal RNA. [2]

RNA isolation from recalcitrant plant tissue

The isolation of high-quality RNA from various tissues (leaves, pedicels, glandular trichomes) of garden geranium (Pelargonium xhortorum) using various published methods is difficult due to numerous oxidizing compounds. A new RNA extraction method was developed through the combination and modification of two separate procedures (Rochester et al., 1986; Manning 1991). In addition to geranium tissues, this method is successful when used with other recalcitrant tissues such as mature needles of white pine (Pinus strobus) and mature leaves of poinsettia (Euphorbia pulcherrima). RNA quality was judged by spectrophotometric readings, denaturing agarose gels, and successful reverse transcription. [3]

Isolation, Cloning and Co-expression of Hepatitis C Virus Envelope Proteins: As Potential HCV Detecting Antigens

Aim: Approximately 3% of the world population is infected with Hepatitis C virus (HCV) which is the main cause of chronic liver disease. Blood transfusion is thought to be the leading cause of global epidemic of HCV. The envelope proteins E1 and E2 are involved in the early stages of the virus life cycle. These proteins have a major role in binding to receptors on the cell surface, fusion and integration of the virus into the host cell. Considering the potency of E1 and E2 in the development of diagnostic methods, the aim of our present study was co-expression of recombinant envelope proteins in eukaryotic HEK293 (human embryonic kidney) cells.

Methods: The viral genomic RNA was used for cDNA (complementary DNA) synthesis. Isolation of HCV envelope proteins coding fragment was performed using cDNA and specific primers. The target gene was cloned into pcDNA3.1 expression vector, and transfected into HEK293 cells, an expression host. Accuracy of the cloning and expression was confirmed using PCR and Western blot analysis.

Results: The isolation and cloning of the gene fragment encoding the E1 and E2 proteins was successful. Co-expression of these proteins was confirmed using monoclonal antibodies specific for each protein.

Conclusion: This study showed that HEK293 host cell is suitable for the expression of hepatitis C virus E1 and E2 coding gene. These proteins can be used in numerous virological studies and detection of HCV infection. [4]

Isolation and Molecular Characterization of Lactic Acid Bacteria Isolated from Fresh Fruits and Vegetables Using Nested PCR Analysis

Aims: The study investigated the diversity and identities of Lactic Acid Bacteria (LAB) isolated from different fresh fruits and vegetables using Molecular Nested PCR analysis with the view of identifying LAB with anti-microbial potentials.

Study Design: Nested PCR approach was used in this study employing universal 16S rRNA gene primers in the first round PCR and LAB specific Primers in the second round PCR with the view of generating specific Nested PCR products for the LAB diversity present in the samples.

Place and Duration of Study: Biotechnology Centre of Federal University of Agriculture, Abeokuta, Ogun State, Nigeria, between January 2011 and February 2012.

Methodology: Forty Gram positive, catalase negative strains of LAB were isolated from fresh fruits and vegetables on Man Rogosa and Sharpe agar (Lab M) using streaking method. Standard molecular methods were used for DNA extraction (Norgen Biotek kit method, Canada), Polymerase Chain Reaction (PCR) Amplification, Electrophoresis, Purification and Sequencing of generated Nested PCR products (Macrogen Inc., USA).

Results: The partial sequences obtained were deposited in the database of National Centre for Biotechnology Information (NCBI). Isolates were identified based upon the sequences as Weissella cibaria (5 isolates, 27.78%), Weissella kimchi (5, 27.78%), Weissella paramensenteroides (3, 16.67%), Lactobacillus plantarum (2, 11.11%), Pediococcus pentosaceus (2, 11.11%) and Lactobacillus pentosus (1, 5.56%) from fresh vegetable; while Weissella cibaria (4, 18.18%), Weissella confusa (3, 13.64%), Leuconostoc paramensenteroides (1, 4.55%), Lactobacillus plantarum (8, 36.36%), Lactobacillus paraplantarum (1, 4.55%) and Lactobacillus pentosus (1, 4.55%) were identified from fresh fruits.

Conclusion: This study shows that potentially LAB can be quickly and holistically characterized by molecular methods to specie level by nested PCR analysis of the bacteria isolate genomic DNA using universal 16S rRNA primers and LAB specific primer. [5]

Reference

[1] Chomczynski, P. and Sacchi, N., 2006. The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nature protocols, 1(2), pp.581-585.

[2] Eldh, M., Lötvall, J., Malmhäll, C. and Ekström, K., 2012. Importance of RNA isolation methods for analysis of exosomal RNA: evaluation of different methods. Molecular immunology, 50(4), pp.278-286.

[3] Schultz, D.J., Craig, R., Cox-Foster, D.L., Mumma, R.O. and Medford, J.I., 1994. RNA isolation from recalcitrant plant tissue. Plant Molecular Biology Reporter, 12(4), pp.310-316.

[4] Mohammadipour, M., Ahangari, G. and Sadeghizadeh, M. (2015) “Isolation, Cloning and Co-expression of Hepatitis C Virus Envelope Proteins: As Potential HCV Detecting Antigens”, Journal of Advances in Medicine and Medical Research, 9(11), pp. 1-8. doi: 10.9734/BJMMR/2015/16720.

[5] Emerenini, E. C., Afolabi, O. R., Okolie, P. I. and Akintokun, A. K. (2013) “Isolation and Molecular Characterization of Lactic Acid Bacteria Isolated from Fresh Fruits and Vegetables Using Nested PCR Analysis”, Microbiology Research Journal International, 3(3), pp. 368-377. doi: 10.9734/BMRJ/2013/2520.

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