Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria
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Using existing protocols, RNA extracted from seeds rich in starch often results in poor quality RNA, making it inappropriate for downstream applications. Though some methods are proposed for extracting RNA from plant tissue rich in starch and other polysaccharides, they invariably yield less and poor quality RNA. In order to obtain high yield and quality RNA from seeds and other plant tissues including roots a modified SDS-LiCl method was compared with existing methods, including TRIZOL kit (Invitrogen), Plant RNeasy mini kit (Qiagen), Furtado (2014) method, and CTAB-LiCl method. Modifications in the extraction buffer and solutions used for RNA precipitation resulted in a robust method for extracting RNA in seeds and roots, where extracting quality RNA is challenging. The modified SDS-LiCl method revealed intense RNA bands through gel electrophoresis and a nanodrop spectrophotometer detected ratios of ≥ 2 and 1.8 for A260/A230 and A260/A280, respectively. The absence of starch co-precipitation during RNA extraction resulted in enhanced yield and quality of RNA with RIN values of 7-9, quantified using a bioanalyzer. The high-quality RNA obtained was demonstrated to be suitable for downstream applications, such as cDNA synthesis, gene amplification, and RT-qPCR. The method was also effective in extracting RNA from seeds of other cereals including field-grown sorghum and corn. The modified SDS-LiCl method is a robust and highly reproducible RNA extraction method for plant tissues rich in starch and other secondary metabolites. The modified SDS-LiCl method successfully extracted high yield and quality RNA from mature, developing, and germinated seeds, leaves, and roots exposed to different abiotic stresses.
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Renal cortex
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genomic DNA
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Total RNA was isolated from Dendrobium displaying different status by using the methods of TRIZOL Regent, Guanidine Thiocyanate, Tris-H_3BO_4, and improved RNA isolation. The quality of total RNA was analyzed through gel electrophoresis and UV spectrometer. The results indicated that the RNA isolated by the improved RNA isolation method showed clear bands of 28 S rRNA and 18 S rRNA,and the value of OD_260 nm/OD_280 nm was close to 2.0. RNA isolated by the other three methods degraded and dispersed in some degrees.RNA isolated by the improved RNA isolation method could be reverse to cDNA. The cDNA was used for RAPD amplifying and two clear bands could be observed in agarose gel. These results demonstrate that the quality and purity of the RNA obtained by the improved RNA isolation method can meet the demands of molecular biology experiment.
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Agarose gel electrophoresis
Dendrobium
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Transcriptomic information provides fundamental insights into biological processes. Extraction of quality RNA is a challenging step, and preservation and extraction protocols need to be adjusted in many cases. Our objectives were to optimize preservation protocols for isolation of high-quality RNA from diverse echinoderm tissues and to compare the utility of parameters as absorbance ratios and RIN values to assess RNA quality. Three different tissues (gonad, oesophagus and coelomocytes) were selected from the sea urchin Arbacia lixula. Solid tissues were flash-frozen and stored at -80 °C until processed. Four preservation treatments were applied to coelomocytes: flash freezing and storage at -80 °C, RNAlater and storage at -20 °C, preservation in TRIzol reagent and storage at -80 °C and direct extraction with TRIzol from fresh cells. Extractions of total RNA were performed with a modified TRIzol protocol for all tissues. Our results showed high values of RNA quantity and quality for all tissues, showing nonsignificant differences among them. However, while flash freezing was effective for solid tissues, it was inadequate for coelomocytes because of the low quality of the RNA extractions. Coelomocytes preserved in RNAlater displayed large variability in RNA integrity and insufficient RNA amount for further isolation of mRNA. TRIzol was the most efficient system for stabilizing RNA which resulted on high RNA quality and quantity. We did not detect correlation between absorbance ratios and RNA integrity. The best strategies for assessing RNA integrity was the visualization of 18S rRNA and 28S rRNA bands in agarose gels and estimation of RIN values with Agilent Bioanalyzer chips.
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Isolation of high-quality RNA from seeds is very critical for seed specific gene analysis. An investigation and comparison on the use of CTAB, SDS and TRIzol extraction procedures to extract high-quality RNA from grains of medical njavara rice were carried out in the present study. These protocols either failed to yield RNA or resulted in reduced yield with poor quality of RNA from Njavara rice grains. The starch and secondary metabolites present in njavara rice seeds hindered the isolation and the resuspension of precipitated RNA or contaminated the RNA pellets by co-precipitation. Hence, we modified the TRIzol RNA extraction protocol by addition of 0.5% N-lauryl sarcosine, 2% β-Mercaptoethanol and 1% PVP. Highly pure (A260/A280 ratio ranged from 1.9 to 2.0) and intact RNA with a higher yield (up to 500µg/ml of RNA) could be obtained using modified TRIzol RNA extraction protocol. RNA obtained was testified and efficiently utilized for the cDNA preparation and amplification of Ubiquitin gene.
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Objective To study the impacts of different sample storage conditions. on RNA isolation efficiency and RNA integrity. Methods Total RNA was isolated from peripheral blood samples (179) stored at different temperatures with different durations. Twenty blood samples were used to investigate the impacts from sample freezing and thawing. Total RNA concentration, purity and integrity were tested. Seventy-three of 179 RNA samples were re-tested, after stored at-80℃ for 1 week, with the data compared to the primarily tested results. Results RNA concentration from fresh blood was the highest 306.51ng/μl on average. Samples after two cycles of freeze/thaw were still available for sufficient RNA isolation, with an average concentration of 181.98 ng/μl, but the integrity of RNA decreased slightly. Both the concentration and purity of RNA declined slightly after 1-week storage at-80℃. Conclusions Blood sample storage condition has significant impacts on RNA isolated concentration. Trizol method is quite stable to isolate sufficient amount RNA from small volume of blood. RNA sample degenerates slightly at-80℃ even for a short time.
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ABSTRACT Current RNA purification methods widely use silica-based columns that allow quick isolation of high-quality and good quantities of RNA. However, the major limitations include high cost, the requirement of different kits for small RNA isolation, genomic DNA contamination, and not being flexible. Here, we used the in-house RNA isolation reagent (RIR) for cell lysis, followed by RNA precipitation using isopropanol. RNA isolated using the in-house RIR resulted in a similar quantity and quality compared to the commercial TRIzol. Furthermore, the commercial RNA isolation kits with silica-based columns recommend genomic DNA digestion during or after RNA purification, adding time and cost to RNA purification. Here, we developed an optimized in-house protocol for isolating high-quality RNA free of genomic DNA contamination using magnetic silica beads without needing DNase digestion. Additionally, our method purifies total RNA along with the small RNA fraction, including miRNAs, which usually require a separate kit for extraction. Additionally, the RNA prepared with our method was equally suitable for mRNA and miRNA expression analysis using RT-qPCR. Together, the in-house method of RNA isolation using the magnetic silica beads has exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.
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genomic DNA
Isolation
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Transcriptional profile of spermatozoa can be used to assess male fertility; differences in spermatozoa attributes require adjustment of protocols for RNA isolation. Ten RNA precipitation modifications of heated TRIzol protocol were applied to total RNA extraction form bull spermatozoa. Motile spermatozoa were isolated from cryopreserved semen straws prepared from an ejaculate of a fertile bull. To evaluate total RNA purity and quantity, Ribogreen RNA detection system and Nanodrop 2000 were used. In RiboGreen assay, a consistent standard curve was not obtained, thus the measurements were ignored. Total RNA purity and concentrations also were assessed using Nanodrop. The A260/280 ratio obtained from Nanodrop showed a good range in the isolated total RNA samples (1.9±0.4). However, all RNA samples presented very low A260/230 ratios (≥1.26). It might be the effect of low concentration of the isolated total RNA. Statistical analysis of total RNA concentrations showed using manufacturer standard protocol had the highest concentration yield. Using either ethanol or Isopropanol as RNA precipitant and changing the incubation temperature had not significant effect on RNA concentration yields. However incubating samples in freezer overnight lowered the total RNA concentration. Using glycogen as a RNA carrier increased the total RNA precipitation yield. In other similar studies the highest total RNA concentration was obtained by using RNA carrier. Validating these findings by qRT-PCR and Bioanalyser might be helpful to enhance the total RNA extraction yield from bull sperm as well as for subsequent genomic studies of bull fertility.
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