Effect of heparin, spermidine, and Be2+ ions on phosphatase and ribonuclease activity of rat liver cell nuclei

Perevoshchikova, K. A.; Prokop, Kh.; Hering, Bernhard J.; Koen, Ya. M.; Zbarskii, I. B.

doi:10.1007/bf00801386

Cited by 6 publications

(2 citation statements)

References 13 publications

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“…The present protocol used 5% β-ME and 0.5 g/l spermidine in the CTAB extraction buffer. β-ME, a potent reducing agent, plays a significant role in the RNA extraction protocols that irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for enzyme functionality and also other contaminating proteins released during tissue disruption and homogenization [40,46,47]. The use of 1-2% β-ME was reported as effective in most protocols [4] but relied on even the tissue types [37].…”

Section: Resultsmentioning

confidence: 99%

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DNA-free high-quality RNA extraction from 39 difficult-to-extract plant species (representing seasonal tissues and tissue types) of 32 families, and its validation for downstream molecular applications

et al. 2023

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Background High-purity RNA serves as the basic requirement for downstream molecular analysis of plant species, especially the differential expression of genes to various biotic and abiotic stimuli. But, the extraction of high-quality RNA is usually difficult from plants rich in polysaccharides and polyphenols, and their presence usually interferes with the downstream applications. The aim of the study is to optimize the extraction of high-quality RNA from diverse plant species/tissues useful for downstream molecular applications. Results Extraction of RNA using commercially available RNA extraction kits and routine hexadecyltrimethylammonium bromide (CTAB) methods did not yield good quality DNA-free RNA from Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera. A reliable protocol for the extraction of high-quality RNA from mature leaves of these difficult-to-extract trees was optimized after screening nine different methods. The DNase I-, and proteinase K treatment-free modified method, consisting of extraction with CTAB method followed by TRIzol, yielded high-quality DNA-free RNA with an A260/A280 and A260/A230 ratios > 2.0. Extraction of RNA from Conocarpus, the most difficult one, was successful by avoiding the heat incubation of ground tissue in a buffer at 65 oC. Pre-warming of the buffer for 5–10 min was sufficient to extract good-quality RNA. RNA integrity number of the extracted RNA samples ranged between 7 and 9.1, and the gel electrophoresis displayed intact bands of 28S and 18S RNA. A cDNA library constructed from the RNA of P. cineraria was used for the downstream applications. Real-time qPCR analysis using the cDNA from P. cineraria RNA confirmed the quality. The extraction of good quality RNA from samples of the desert-growing P. cineraria (> 20-years-old) collected in alternate months of the year 2021 (January to December covering winter, spring, autumn, and the very dry and hot summer) proved the efficacy of the protocol. The protocol’s broad applicability was further validated by extracting good-quality RNA from 36 difficult-to-extract plant species, including tissues such as roots, flowers, floral organs, fruits, and seeds. Conclusions The modified DNase I and Proteinase K treatment-free protocol enables to extract DNA-free, high-quality, intact RNA from a total of 39 difficult-to-extract plant species belonging to 32 angiosperm families is useful to extract good-quality RNA from dicots and monocots irrespective of tissue types and growing seasons.

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Section: Resultsmentioning

confidence: 99%

“…However, in the present study, 5% β-ME was efficient in the case of C. erectus, and we used 5% in all other species. Spermidine plays a significant role as an RNase inhibitor [40,46,48]. RNA degradation depends not only on the presence of RNase but also on poly(A) length and spermidine concentration [47].…”

Section: Resultsmentioning

confidence: 99%

DNA-free high-quality RNA extraction from 39 difficult-to-extract plant species (representing seasonal tissues and tissue types) of 32 families, and its validation for downstream molecular applications

et al. 2023

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Some features of nucleo-cytoplasmic RNA transport from isolated nuclei

1981

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Messenger RNA is released preferentially from isolated rat liver nuclei in the presence of the ATP-generating system and cytosol. The release is suppressed by spermidine, while cytoplasmic RNase inhibitor was ineffective and PCMB like some other thiol-blocking agents inhibitory. Cytoplasmic SOD added to the system strongly suppressed RNA release. A similar effect could be obtained by anaerobiosis due to addition of SMP. In both cases the inhibition is reversed by cyanide. In contrast to normal liver where the generation of superoxide radicals takes place almost exclusively in microsomes and is coupled with the oxidation of NADPH, in mouse ascites hepatoma 22a the generation of superoxide radicals occurs mainly in the nuclear envelope and is coupled wih the oxidation of both NADPH and NADH and inhibited by cyanide.

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The role of protein phosphokinase and protein phosphatase during the nuclear envelope nucleoside triphosphatase reaction

Bachmann

Bernd

Schröder

et al. 1984

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Effect of heparin, spermidine, and Be2+ ions on phosphatase and ribonuclease activity of rat liver cell nuclei

Cited by 6 publications

References 13 publications

DNA-free high-quality RNA extraction from 39 difficult-to-extract plant species (representing seasonal tissues and tissue types) of 32 families, and its validation for downstream molecular applications

DNA-free high-quality RNA extraction from 39 difficult-to-extract plant species (representing seasonal tissues and tissue types) of 32 families, and its validation for downstream molecular applications

Some features of nucleo-cytoplasmic RNA transport from isolated nuclei

The role of protein phosphokinase and protein phosphatase during the nuclear envelope nucleoside triphosphatase reaction

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