Isolation of high-quality RNA from Platycladus orientalis and other Cupressaceae plants

Platycladus orientalis has a lifespan of several thousand years in China, making it a good plant in which to study aging at the molecular level, but this requires sufficient quantities of high-quality P. orientalis RNA. However, no appropriate methods have been reported for total RNA isolation from P. orientalis leaves. The TRIzol method did not extract RNA, while cetyltrimethylammonium bromide, sodium dodecyl sulfate-phenol, and plant RNAout kit (Tianz, Inc., China) protocols resulted in low yields of poor quality RNA. Isolating total RNA using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) resulted in a high-quality product but a low yield. However, the two-step removal of polyphenols and polysaccharides in the improved plant RNAout kit protocol resulted in the isolation of RNA with a 28S:18S rRNA ratio of band intensities that was ~2:1, the A260/A280 absorbance ratio was 2.03, and the total RNA yield from P. orientalis leaves was high. This protocol was tested on different P. orientalis tissues of different ages and on leaves of five other Cupressaceae plants. The total RNAs were successfully used in complementary DNA synthesis for transcriptome sequencing and would be suitable to use in additional experiments. The results of this study will benefit future studies in Cupressaceae plants.

Cloning and expression analysis on arginine decarboxylase genes in Atropa belladonna / 中草药

Objective: To clone the full-length cDNAs encoding arginine decarboxylase (ADC) from Atropa belladonna, and to characterize the genes at the bioinformatics and expression levels. Methods: cDNAs were used as templates, the full-length cDNAs of ADC in A. belladonna was cloned through rapid amplification of cDNA ends (RACE) technique; Bioinformatics analysis of AbADC genes was performed by BLAST and PBIL on line. The expression levels of the two AbADC genes in different tissues as well as under two different types of stresses were detected based on qPCR analysis. Results: Two ADC genes, namely AbADC1 and AbADC2, were cloned from A. belladonna. AbADC1 was 2 817 bp in length that encoded 712 amino acids with the highest identity of 89% with ADC from Solanum tuberosum; The full-length cDNA of AbADC2 was 2 992 bp in length that encoded 715 amino acids with the highest identity of 90% with ADC from Datura stramonium. The expression level of AbADC1 was significantly higher in secondary roots than that in any other organ, while the AbADC2 expression level was higher in main roots than that in other detected organs. The transcriptional levels of both AbADC1 and AbADC2 were not affected under salinity stress; AbADC2 expression decreased under cold stress, while AbADC1 did not. Conclusion: The cloning and characterization of the cDNAs encoding ADC from A. belladonna are reported for the first time, which provides the new candidate genes for engineering biosynthetic pathway of tropane alkaloids.

Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani

Background Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is one of the most important pathogens of rice (Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani, five methods, including E.Z.N.A.™ Fungal RNA Kit, sodium dodecyl sulfate (SDS)-sodium borate, SDS-polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study. Results The electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS-sodium borate, SDS-PVP and E.Z.N.A.™ Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment. Conclusion It is concluded that SDS-sodium borate and SDS-PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A.™ Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield.

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of β-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues.

The Effect of Nitrous Oxide and Isoflurane on the Total RNA Yield from the Cochlea of the Rats / 华中科技大学学报（医学）（英德文版）

The possible mechanism of inhalation anesthetics on the internal auditory impairment of the rat was investigated by determining the effect of nitrous oxide (N20) and isoflurane on the total RNA yield from the cochlea of the rats. Thirty healthy Wistar rats were randomly divided into 3 groups: group C (control group, n=10) with a 3-h unremitting inhalation of 50% O2 group N (ex-periment group, n= 10) with a continuous inhalation of 50% N2O+50% O2for 3 h, and group I (ex-periment group, n=10) with a 3-h sustained inhalation of 2.5% isoflurane. The TRIzol in combination with RNeasy was used to respectively extract the total RNA from cochlea of rats in the 3 groups. Spectrophotometry was used to detect total RNA yield and electrophoresis to detect the quality. The total RNA extracted from the cochlea of the rats in the groups C and N was 7.69 and 6.51 μg, respec- tively. There was a 15% decrease in the N group as compared with group C. The total RNA from the rats in the group I was 7.32 μg, and there was hardly any change in the group as compared with the group C. The value of A260/A280 in groups C, N and I was 2.07, 2.04 and 2.04, respectively, showing a very high RNA purity. The result of gel electrophoresis suggested that there was no degradation in the total RNA. It was suggested that the interference of N2O on the cochlear RNA yield might be one of the reasons which cause an injury of the ear. The isoflurane shows no harm on the heating.

A method with TRIzol~ reagent and liquid nitrogen to extract high-quality RNA from rat pancreas / 西安交通大学学报(医学版)

Objective To establish a quick,economical and reproducible method for high-quality RNA extraction from pancreas.Methods We utilized TRIzol Reagent and liquid nitrogen to isolate total RNA from the rat pancreas.The RNA quality was determined by detection of its content and optic density(A) at 260/280nm,and electrophoresis in 1% non-denatured agarose gel.Then reverse transcription-polymerase chain reaction(RT-PCR) was performed to detect expression of the pancreas-specific genes.Results The content of the total RNA extracted from the rat pancreas reached 3-6?g/mg pancreatic tissues,and A260/280 ratio was 1.75-1.89.Electrophoresis of the total RNA showed 28S and 18S rRNA bands with clear smear between them.The RT-PCR products of pancreas-specific genes including insulin 1,glucagon,?-amylase and housekeeping gene ?-actin all exhibited clear bands on 1% agarose gel,which were located in the expected positions,respectively.Conclusion These results suggest that we have successfully isolated the high-quality and intact RNA from the rat pancreas with TRIzol Reagent and liquid nitrogen.The extracted total RNA can be used in RT-PCR for pancreatic gene expression.

An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material

We have developed an improved method for constructing a full-length cDNA library using small quantity of material by modifying the original oligo-capping method. In our devised method, total RNAs are used in sequential oligo-capping steps directly without preliminary mRNA purification. Using this method, we constructed full- length cDNA libraries from 100 microg of total RNA. These libraries contained 8x10(5) to 8x10(6) independent clones with average insert sizes of 2.0 kb. Moreover, the number of full-length cDNAs containing the translation initiation codon ATG in the constructed libraries was estimated to 60-70%. In addition, 54% of the known cDNAs had a longer 5' end than the corresponding genes in the public database. Our results show that the method can be effectively used to construct full-length enriched cDNA libraries, especially, if starting material is limited.

Induction of Cytotoxic T Lymphocytes by Dendritic Cells Pulsed with Murine Leukemic Cell RNA / 대한혈액학회지

BACKGROUND: This study was done to assess the feasibility of dendritic cell generation from murine bone marrow and the efficacy of dendritic cells pulsed with total RNA to induce specific cytotoxic T lymphocyte response against leukemic cells. METHODS: Nucleated cells of inbred BALB/c mice were obtained and cultured with granulocyte/macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) to induce dendritic cells. Total RNA of WEHI-3BD+, a myelomonocytic leukemia cell line from BALB/c, was transfected into the dendritic cells using liposome. RNA pulsed dendritic cells were irradiated and administered to the BALB/c mice intraperitoneally and splenic T lymphocytes were harvested. After restimulation with leukemic cells, T cell proliferation and specific cytotoxicity was assessed. RESULTS: Cells cultured with GM-CSF and lipopolysaccaride were found to have prominent dendritic processes. The percentage of cells showing high expression of both MHC class II and CD80, CD86, or CD11c was 69.6 %, 63.7%, and 41.8%, respectively. T cells stimulated by WEHI-3BD+ total RNA pulsed dendritic cells using DOTAP showed enhanced proliferation than those stimulated by total RNA or media only (P=0.05). When T cells were cocultured with WEHI-3BD+ as target cells, T cells stimulated by WEHI-3BD+ total RNA pulsed dendritic cells using DOTAP showed much increased cytotoxicity than controls. CONCLUSION: Dendritic cells pulsed with total leukemic RNA could stimulate T cells to induce specific cytotoxic effect.

IMPACT OF FRACTIONED IR RADIATION ON TOTAL RNA AND MDR_1 GENE OF NCI-H446 CELL LINE / 解放军医学杂志

To study the effects of fractioned ir radiation on the total RNA and MDR 1 mRNA of NCI H 446 small cell lung cancer cell line, NCI H446 cells in the period of exponential growth were exposed to 60 Co ? radiation at 2 Gy/fraction, 2 fraction/week, with the cumulative dose of 50 Gy by 25 fractions. The total RNA of the cells of the irradiated group and control group were extracted by the acid guanidine thiocyanate phenol chloroform method and the amount of the expression of MDR 1 mRNA was assessed by qualitative RT PCR assays. Under the conditions of same cell number and volume, in cells of the control group, the concentration of the total RNA was 25 9 ?g/ml and the ratio of MDR 1 DNA/? actinDNA was 1 078, whereas in cells of the irradiation group, the corresponding values were 16 6 ?g/ml and 1 338, respectively. It is concluded that, for the NCI H446 small cell lung cancer cell line, a high dose accumulated during fractioned irradiation can inhibit the synthesis of its total RNA and enhance the expression of its MDR 1 gene at the same time.

In vitro induction of p210~(Bcr-Abl) protein-specific cytotoxic T cell responses using 562 cell total RNA transfected human dendritic cells / 中国输血杂志

Objective To investigate whether human monocyte-derived dendritic cells (DCs) can express p210 Bcr-Abl protein and induce antigen-specific CTL responses in vitro after transfection with total RNA of K562 cells (K562-RNA).Methods Immature DCs were derived from human peripheral blood monocytes after 5 day incubation in the presence of GM-CSF and IL-4. The cells were then transfected with K562-RNA using electroporation or DOTAP lipofection. To verify the successful transfection of DCs with K562-RNA, Bcr-Abl fusion gene expression was determined by RT-PCR and Western blot. The immune phenotypes of the DCs were analyzed by flow cytometry. CTL cytotoxicity was assayed by propidium iodide (PI) stain and flow cytometry. The amount of DCs, CD1a expression and purity of DCs were measured by FACS.Results Bcr-Abl fusion gene appeared in the DCs after transfection with K562 cell total RNA. But 24 hours later, the Bcr-Abl mRNA from the K562-RNA transfected DCs disappeared, while the cells were expressing p210 Bcr-Abl protein. The transfected DCs could significantly promote T lymphocytes to kill the target K562 cells. We found that PBMC can be induced to DC in culture medium containing human plasma, suggesting a potential for clinical application.Conclusion Human dendritic cells transfected by K562 total RNA can induce effective p210 Bcr-Abl protein-specific immune responses, which might broaden the spectrum of possible DC-based clinical applications.