RNA isolation from high-phenolic freeze-dried tea (Camellia sinensis) leaves

Jaiprakash, M.; Pillai, Beena; Venkatesh, Purna; Subramanian, Nithya; Sinkar, Vilas P.; Sadhale, Parag P.

doi:10.1007/bf02772599

Cited by 20 publications

(30 citation statements)

References 4 publications

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“…Agarose-formaldehyde gel electrophoresis resolved the two ribosomal RNA subunits (28S and 18S), showing intact RNA. As reported by others, no effect of the lyophilization process was found in this study [61,62]. Also, we showed that there was no effect of elicitation treatment on RNA integrity.…”

Section: Resultssupporting

confidence: 76%

Selection of reference genes for qPCR in hairy root cultures of peanut

et al. 2011

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BackgroundHairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments.ResultsA total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc).ConclusionsThis work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.

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Section: Resultssupporting

confidence: 76%

Selection of reference genes for qPCR in hairy root cultures of peanut

et al. 2011

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“…Different letters mean a significant difference (p b 0.05) between treatments. activity of proteolytic enzymes and nucleases, diminishing oxidation and cellular component degradation [7]. According to the RIN values in all the samples evaluated, we report that RNA obtained from these tissues has good integrity [13].…”

Section: Resultsmentioning

confidence: 99%

“…Lyophilization has been widely used for the freeze-drying and storage of various biological samples in the food industry, pharmacy biotechnology and tissue engineering. Despite the advantages that this tool offers, RNA extraction from lyophilized tissues, such as mouse tissues [6], tea leaves [7], tuber and root tissues, such as potato, turnip, sweet potato and radish [5], has not been extensively reported. Saha et al [8] reported the disadvantage that lyophilized cotton tissue had total RNA low quality.…”

Section: Introductionmentioning

confidence: 99%

“…Saha et al [8] reported the disadvantage that lyophilized cotton tissue had total RNA low quality. Theoretically, lyophilization should limit or delay cellular component degradation by inactivation of proteolytic enzymes and nucleases [7,8], allowing long-term room temperature storage or long-distance transportation if the seal is maintained. In the present study, we evaluated the effects of the freeze-dried process using dormant grapevine buds on the quality of the obtained RNA that will be used for further cDNA synthesis, amplification and gene expression in transcriptomic contexts.…”

Section: Introductionmentioning

confidence: 99%

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Total RNA quality of lyophilized and cryopreserved dormant grapevine buds

García-Baldenegro

Vargas‐Arispuro

Islas-Osuna

et al. 2015

Electronic Journal of Biotechnology

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a b s t r a c tBackground: Plant tissues must be preserved in their collection state, especially for genome-wide expression profile studies. Lyophilization is a feasible, affordable tool when fresh tissues cannot be shipped at ultralow temperatures from their origin to the place of analysis. In this study, the total RNA quality of dormant grapevine buds (Vitis vinifera L. cv. 'Flame Seedless') of freeze-dried samples stored at room temperature conditions was evaluated and compared to that of cryopreserved (-80°C) grapevine buds. Results: Good yield and quality of RNA were obtained from freeze-dried dormant buds stored at room temperature for 0, 3 and 6 weeks after they were lyophilized. Further experiments confirmed that the extracted total RNA could be used for actin and β-tubulin PCR gene amplification. Conclusion: High-quality RNA that is useful for downstream applications was obtained from freeze-dried dormant grapevine bud tissue, similarly to the RNA obtained from cryopreserved dormant grapevine buds.

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“…A potential application of lyophilization which has been exploited in plant tissue [2,3] but not yet explored in animal tissue is whether removing the water from traditionally difficult to homogenize tissues (i.e. bone, gill, gut and eye) allows the sample to be more easily managed.…”

Section: Discussionmentioning

confidence: 99%