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Isolation of High-Quality Total RNA from Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook).

Ma Z, Huang B, Xu S, Chen Y, Li S, Lin S - PLoS ONE (2015)

Bottom Line: RNA isolations were performed within two hours, RNA quality was measured for yield and purity.Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing.It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China; State Forestry Administration Engineering Research Center of Chinese Fir, Fuzhou, China.

ABSTRACT
RNA isolation with RNA in a high quantity is a basic analytical method in plant genetics, molecular biology and related physiological investigations. To understand the genetic and molecular biology of Chinese fir, sufficient high-quality total RNA must be obtained for cDNA library construction and other downstream molecular applications. However, extracting RNA from Chinese fir is difficult and often requires the modification of existing protocols. Chinese fir tissues containing large amounts of polysaccharides and polyphenol compounds and are one of the most difficult plant tissues for RNA isolation. Therefore, we developed a simple method for extracting high-quality RNA from Chinese fir tissues. RNA isolations were performed within two hours, RNA quality was measured for yield and purity. Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing. It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications. Thus, this protocol represents a simple, efficient, and low-cost method.

No MeSH data available.


Example of agarose gel electrophoresis of total RNA isolated.Visualization of three intact RNA bands for 28 S RNA (b), 18 S RNA (c) and 5 S RNA (d). Lanes A and B contain 1 μg of total RNA from Chinese fir leaves, and Lanes C and D contain 1 μg of total RNA from Chinese fir roots.
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pone.0130234.g001: Example of agarose gel electrophoresis of total RNA isolated.Visualization of three intact RNA bands for 28 S RNA (b), 18 S RNA (c) and 5 S RNA (d). Lanes A and B contain 1 μg of total RNA from Chinese fir leaves, and Lanes C and D contain 1 μg of total RNA from Chinese fir roots.

Mentions: The quality of isolated total RNA described in this study was confirmed in several ways. Firstly, 1.0% agarose gel electrophoresis showed that ribosomal bands of 28 S (b), 18 S (c) and 5 S (d) were intact and bright, demonstrating that the total RNA isolated with our detailed method were not degraded, and no high molecular weight gDNA contamination was detected (Fig 1 and S1 Fig). In addition, the ratio of 28 S and 18 S ranged from 1.4 to 2.1, indicating good RNA quality (Table 1). Secondly, spectrophotometer analysis showed that the A260/280 ratios of RNA samples ranged between 1.9 and 2.1 (Table 1), indicating that the RNA extracted by this approach was largely free of contaminating proteins, DNA, polyphenolics, and polysaccharides. In addition, the A260/230 values were ranged from 1.82 to 2.18 (Table 1), indicating that RNA was of high purity and without contamination by organic solvents, secondary metabolites, polyphenolics and polysaccharides [4]. Thirdly, the quality of total RNA was assessed using RT-PCR. RNA samples were successfully reverse transcribed using Invitrogen SuperScript IIReverse Transcriptase (Life Technologies, USA), and the target gene was amplified from cDNA by PCR (Fig 2). As indicated in Fig 2, the PCR products were checked for the presence of the objective band on 1% agarose gel. PCR products for detecting the ALMT1 gene exhibit the target band with the expected size of approximately 600 bp (Fig 2). Last but perhaps most importantly, an electropherogram of the total RNA isolated from Chinese fir leaves and roots tissues was generated after analysis with the Agilent 2100 Bioanalyzer. A representative electropherogram of total RNA and the generated gel images showed clear peaks of rRNAs and intact RNA of high quality (Fig 3). In general, RNA with a value above 7.0 is required to produce good results in a next-generation sequencing analysis [4]. The RIN values are shown for three samples isolated with our detailed method (Table 1 and Fig 3). We obtained the total RNA of leaf and root tissues of Chinese fir with RINs of 7.8 (Fig 3A), 9.1 (Fig 3B) and 9.2 (Fig 3C), respectively, indicating no degradation of the RNA, and the Bioanalyzer results of the residual samples were addressed in supplement files (S2 Fig, S3 Fig, S4 Fig, S5 Fig and S6 Fig). These RIN values are much higher than those in previously published reports [20]. The high-integrity RNA obtained from the different Chinese fir tissues used in the current method was suitable for RT-PCR and construction of cDNA libraries.


Isolation of High-Quality Total RNA from Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook).

Ma Z, Huang B, Xu S, Chen Y, Li S, Lin S - PLoS ONE (2015)

Example of agarose gel electrophoresis of total RNA isolated.Visualization of three intact RNA bands for 28 S RNA (b), 18 S RNA (c) and 5 S RNA (d). Lanes A and B contain 1 μg of total RNA from Chinese fir leaves, and Lanes C and D contain 1 μg of total RNA from Chinese fir roots.
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Related In: Results  -  Collection

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pone.0130234.g001: Example of agarose gel electrophoresis of total RNA isolated.Visualization of three intact RNA bands for 28 S RNA (b), 18 S RNA (c) and 5 S RNA (d). Lanes A and B contain 1 μg of total RNA from Chinese fir leaves, and Lanes C and D contain 1 μg of total RNA from Chinese fir roots.
Mentions: The quality of isolated total RNA described in this study was confirmed in several ways. Firstly, 1.0% agarose gel electrophoresis showed that ribosomal bands of 28 S (b), 18 S (c) and 5 S (d) were intact and bright, demonstrating that the total RNA isolated with our detailed method were not degraded, and no high molecular weight gDNA contamination was detected (Fig 1 and S1 Fig). In addition, the ratio of 28 S and 18 S ranged from 1.4 to 2.1, indicating good RNA quality (Table 1). Secondly, spectrophotometer analysis showed that the A260/280 ratios of RNA samples ranged between 1.9 and 2.1 (Table 1), indicating that the RNA extracted by this approach was largely free of contaminating proteins, DNA, polyphenolics, and polysaccharides. In addition, the A260/230 values were ranged from 1.82 to 2.18 (Table 1), indicating that RNA was of high purity and without contamination by organic solvents, secondary metabolites, polyphenolics and polysaccharides [4]. Thirdly, the quality of total RNA was assessed using RT-PCR. RNA samples were successfully reverse transcribed using Invitrogen SuperScript IIReverse Transcriptase (Life Technologies, USA), and the target gene was amplified from cDNA by PCR (Fig 2). As indicated in Fig 2, the PCR products were checked for the presence of the objective band on 1% agarose gel. PCR products for detecting the ALMT1 gene exhibit the target band with the expected size of approximately 600 bp (Fig 2). Last but perhaps most importantly, an electropherogram of the total RNA isolated from Chinese fir leaves and roots tissues was generated after analysis with the Agilent 2100 Bioanalyzer. A representative electropherogram of total RNA and the generated gel images showed clear peaks of rRNAs and intact RNA of high quality (Fig 3). In general, RNA with a value above 7.0 is required to produce good results in a next-generation sequencing analysis [4]. The RIN values are shown for three samples isolated with our detailed method (Table 1 and Fig 3). We obtained the total RNA of leaf and root tissues of Chinese fir with RINs of 7.8 (Fig 3A), 9.1 (Fig 3B) and 9.2 (Fig 3C), respectively, indicating no degradation of the RNA, and the Bioanalyzer results of the residual samples were addressed in supplement files (S2 Fig, S3 Fig, S4 Fig, S5 Fig and S6 Fig). These RIN values are much higher than those in previously published reports [20]. The high-integrity RNA obtained from the different Chinese fir tissues used in the current method was suitable for RT-PCR and construction of cDNA libraries.

Bottom Line: RNA isolations were performed within two hours, RNA quality was measured for yield and purity.Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing.It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China; State Forestry Administration Engineering Research Center of Chinese Fir, Fuzhou, China.

ABSTRACT
RNA isolation with RNA in a high quantity is a basic analytical method in plant genetics, molecular biology and related physiological investigations. To understand the genetic and molecular biology of Chinese fir, sufficient high-quality total RNA must be obtained for cDNA library construction and other downstream molecular applications. However, extracting RNA from Chinese fir is difficult and often requires the modification of existing protocols. Chinese fir tissues containing large amounts of polysaccharides and polyphenol compounds and are one of the most difficult plant tissues for RNA isolation. Therefore, we developed a simple method for extracting high-quality RNA from Chinese fir tissues. RNA isolations were performed within two hours, RNA quality was measured for yield and purity. Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing. It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications. Thus, this protocol represents a simple, efficient, and low-cost method.

No MeSH data available.