Evaluation of different RNA extraction methods for high-quality total RNA and mRNA from Erwinia amylovora in planta

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

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Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870501700609 ◽

2005 ◽

Vol 17 (6) ◽

pp. 574-578 ◽

Cited By ~ 41

Author(s):

Ming Y. Deng ◽

He Wang ◽

Gordon B. Ward ◽

Tammy R. Beckham ◽

Thomas S. McKenna

Keyword(s):

Real Time ◽

Classical Swine Fever Virus ◽

Rna Extraction ◽

Classical Swine Fever ◽

Extraction Methods ◽

Rt Pcr ◽

Total Rna ◽

Reverse Transcription Pcr ◽

Rna Extraction Methods ◽

Positive Results

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding β-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

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Comparative assessment of four RNA extraction methods and modification to obtain high-quality RNA from Parthenium hysterophorus leaf

3 Biotech ◽

10.1007/s13205-017-1003-3 ◽

2017 ◽

Vol 7 (6) ◽

Cited By ~ 6

Author(s):

Javed Ahmad ◽

M. Affan Baig ◽

Arlene A. Ali ◽

Asma Al-Huqail ◽

M. M. Ibrahim ◽

...

Keyword(s):

Rna Extraction ◽

Extraction Methods ◽

Comparative Assessment ◽

Parthenium Hysterophorus ◽

High Quality ◽

Rna Extraction Methods

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Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems

Genetics and Molecular Research ◽

10.4238/gmr.15017364 ◽

2016 ◽

Vol 15 (1) ◽

Cited By ~ 3

Author(s):

D.V. Silva ◽

S.M.J. Branco ◽

I.S.A. Holanda ◽

S. Royaert ◽

J.C. Motamayor ◽

...

Keyword(s):

Theobroma Cacao ◽

Comparative Evaluation ◽

Rna Extraction ◽

Extraction Methods ◽

Total Rna ◽

Apical Meristems ◽

Shoot Apical Meristems ◽

Rna Extraction Methods ◽

Total Rna Extraction

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Evaluation of Different RNA Extraction Methods from Agropatch Suppressor Assay for Small Quantities of Plant Tissue and Their Application for Analysis of Gene Expression

Notulae Scientia Biologicae ◽

10.15835/nsb10310307 ◽

2018 ◽

Vol 10 (3) ◽

pp. 348-353

Author(s):

Amir G. SHAHRIARI ◽

Aminallah TAHMASEBI

Keyword(s):

Gene Expression ◽

Plant Tissue ◽

Target Genes ◽

Rna Extraction ◽

Extraction Methods ◽

Total Rna ◽

Expression Studies ◽

Rna Extraction Methods ◽

Gene Expression Studies ◽

Commercial Kit

The agroinfiltration assay provides fast and efficient way to transiently express genes into plant cells by Agrobacterium tumefaciens. Extraction of RNA of high quality and sufficient amounts is prerequisite for gene expression studies such as quantitative Real Time PCR (q-PCR) from infiltrated areas in agropatch suppressor assay with small quantities of plant tissue. To attain prime RNA extraction from small tissues of infiltrated N. benthamiana plants with Potato virus A helper component proteinase viral suppressor protein, the efficiency of three RNA extraction methods (LiCl, TRIzol reagent and commercial kit) was evaluated. The total RNA yield with LiCl method was 2.83 and 33.2-fold greater than that of TRIzol reagent and commercial kit, respectively. Also, total RNA yield using TRIzol reagent was 11.7-fold higher than that with commercial kit. The A260/A280 ratio mean for TRI reagent (1.95) and kit (1.9) extractions were within the optimum range.q-PCR revealed that the cycle threshold values of housekeeping gene, EIF-1α and target genes AGO1 and ATG6 for RNA extracted using LiCl and kit were 1.07 to 1.3 and 1.02 to 1.12 times higher than those evaluated with the TRIzol method. Overall, TRIzol method showed the most effective approach for obtaining RNA from N. benthamiana patches in gene expression studies.

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Big data from small tissues: extraction of high-quality RNA for different plant tissue types during oilseed Brassica spp. seed development for RNA-Sequencing

10.1101/2019.12.20.885012 ◽

2019 ◽

Author(s):

Laura Siles ◽

Peter Eastmond ◽

Smita Kurup

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Seed Development ◽

Developmental Stages ◽

Rna Extraction ◽

Rna Isolation ◽

Extraction Methods ◽

High Quality ◽

Gene Expression Analyses ◽

Rna Extraction Methods

AbstractObtaining high-quality RNA for gene expression analyses from different seed tissues is challenging due to the presence of various contaminants, such as polyphenols, polysaccharides and lipids which interfere with RNA extraction methods. At present, the available protocols for extracting RNA from seeds require high amounts of tissue and are mainly focused on extracting RNA from whole seeds. However, extracting RNA at the tissue level enables more detailed studies regarding tissue specific transcriptome during development. Seeds from heart stage embryo to mature developmental stages of Brassica napus and B. oleracea were sampled for isolation of the embryo, endosperm and seed coat tissues. Ovules and gynoecia wall tissue were also collected at the pre-fertilization stage. After testing several RNA extraction methods, E.Z.N.A. Plant RNA Kit and Picopure RNA Isolation kit extraction methods with some modifications, as well as the use of PVPP for seed coats and endosperms at green stages, resulted in high RNA concentrations with clear 28S and 18S bands and high RIN values. Here, we present efficient and reliable RNA extraction methods for different genotypes of Brassica spp for different tissue types during seed development. The high-quality RNA obtained by using these methodologies is suitable for RNA-Sequencing and gene expression analyses.

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