Short Communication Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues

Zhang, Y J; Hao, Xiuhong; Liang, Zongsuo; Ke, Weidong; Guo, Hongbo

doi:10.4238/2013.january.24.14

Cited by 11 publications

(8 citation statements)

References 13 publications

(13 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As a result, the quality (purity and integrity) and yield of isolated total RNA achieved by our method is very high and reproducible irrespective of the species, tissue and physiological state. As compared to a recent improved protocol based on CTAB, phenol and LiCl for preparation of high quality RNA from peach tissues [23], we obtain similar results in quality (A260/A280; A260/A230), but significant improvements in yield. In comparable samples of young tissues (e.g., flower, petal, leaf, stem and fruit), the increase is modest (~1.5-fold); however, the increase is substantial (~10-fold) in adult and stressed tissues of petal, leaf and mature fruit, and even greater (~15-fold) in postharvest storage fruit.…”

Section: Discussionmentioning

confidence: 56%

See 1 more Smart Citation

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

Carriel

Sellés-Marchart

Martínez-Márquez

et al. 2014

Analytical Biochemistry

View full text Add to dashboard Cite

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were>2.0) but also of high yield (up to 720 μg on average [coefficient of variation=21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.

show abstract

Section: Discussionmentioning

confidence: 56%

“…Furthermore, various methods based on cetyltrimethylammonium bromide (CTAB) have been developed for tissues containing high levels of polysaccharides and phenols [19][20][21][22][23];…”

Section: Introductionmentioning

confidence: 99%

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

Carriel

Sellés-Marchart

Martínez-Márquez

et al. 2014

Analytical Biochemistry

View full text Add to dashboard Cite

show abstract

“…TOTAL RNA EXTRACTION AND CDNA SYNTHESIS. Total RNA was extracted from different tissues of N. nucifera according to the cetyltrimethylammonium bromide (CTAB)-LiCl method reported by Zhang et al (2013). One gram of frozen tissue was ground in liquid nitrogen using a mortar and pestle, and quickly transferred to 1 mL extraction buffer prewarmed to 65°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Flower Color Diversity Revealed by Differential Expression of Flavonoid Biosynthetic Genes in Sacred Lotus

Wang

Chen

Yuan

et al. 2016

J. Amer. Soc. Hort. Sci.

View full text Add to dashboard Cite

Sacred lotus (Nelumbo nucifera) is an important aquatic ornamental plant which contains several diverse flower colors, but the underlying mechanisms of its flower coloration remain unclear. In this study, seven complementary DNA (cDNA) clones of genes involved in flavonoid biosynthesis, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), were isolated and characterized. Moreover, expression patterns of these seven genes and pigment profiles were investigated across four N. nucifera cultivars with different flower colors: Zhongguohongbeijing [ZGH (red)], Xinghuafen [XHF (pink)], Molingqiuse [MLQS (yellow)], and Zhufengcuiying [ZFCY (white)]. Real-time quantitative polymerase chain reaction (qRT-PCR) analysis showed that during flower development, transcripts of early biosynthetic genes (NnCHS, NnCHI, and NnF3H) were abundant at the early stage; noticeably, highest expression of NnCHI in MLQS probably induced abundant anthoxanthin synthesis and displayed yellow. Expression of late biosynthetic genes, especially NnDFR and NnANS, was generally consistent with change patterns of anthocyanins in ZGH and XHF, but NnF3′H was barely detectable in the pink cultivars. Meanwhile, negligible expression of NnDFR and NnANS was detected in MLQS and ZFCY, respectively, which blocked their colored anthocyanin biosynthesis. Spatial expression analysis revealed that most flavonoid biosynthetic genes were highly expressed in floral tissues, rather than leaves. These results suggest that in N. nucifera cultivars with different flower colors, flavonoid biosynthesis is differentially regulated by the expression of these flavonoid biosynthetic genes, among which, NnCHI, NnF3′H, NnDFR, and NnANS are supposed to be critical for pigment accumulation, and therefore, affect different flower coloration.

show abstract

“…Protocols for the isolation total RNA from these kinds of plants should be modified individually, because of the interfering role of polyphenolics and polysaccharides. In this regard, there are some modified protocols used to isolate high quality RNA, for example the modified guanidinium phenol chloroform method [43], the hot borate method [44,45], and methods based on the use of CTAB [46][47][48][49][50]. Except for recently modified protocols [51][52][53], some of these are time consuming and fail to provide a high-yield RNA.…”

Section: Rna Extraction From Plantsmentioning

confidence: 99%

Suppression Subtractive Hybridization Versus Next-Generation Sequencing in Plant Genetic Engineering: Challenges and Perspectives

et al. 2015

View full text Add to dashboard Cite

Suppression subtractive hybridization (SSH) is an effective method to identify different genes with different expression levels involved in a variety of biological processes. This method has often been used to study molecular mechanisms of plants in complex relationships with different pathogens and a variety of biotic stresses. Compared to other techniques used in gene expression profiling, SSH needs relatively smaller amounts of the initial materials, with lower costs, and fewer false positives present within the results. Extraction of total RNA from plant species rich in phenolic compounds, carbohydrates, and polysaccharides that easily bind to nucleic acids through cellular mechanisms is difficult and needs to be considered. Remarkable advancement has been achieved in the next-generation sequencing (NGS) field. As a result of progress within fields related to molecular chemistry and biology as well as specialized engineering, parallelization in the sequencing reaction has exceptionally enhanced the overall read number of generated sequences per run. Currently available sequencing platforms support an earlier unparalleled view directly into complex mixes associated with RNA in addition to DNA samples. NGS technology has demonstrated the ability to sequence DNA with remarkable swiftness, therefore allowing previously unthinkable scientific accomplishments along with novel biological purposes. However, the massive amounts of data generated by NGS impose a substantial challenge with regard to data safe-keeping and analysis. This review examines some simple but vital points involved in preparing the initial material for SSH and introduces this method as well as its associated applications to detect different novel genes from different plant species. This review evaluates general concepts, basic applications, plus the probable results of NGS technology in genomics, with unique mention of feasible potential tools as well as bioinformatics.

show abstract

Short Communication Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues

Cited by 11 publications

References 13 publications

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

Flower Color Diversity Revealed by Differential Expression of Flavonoid Biosynthetic Genes in Sacred Lotus

Suppression Subtractive Hybridization Versus Next-Generation Sequencing in Plant Genetic Engineering: Challenges and Perspectives

Contact Info

Product

Resources

About