A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

Many experiments in plant molecular biology require processing of a large number of RNA samples and in some cases large quantities are required for a single application. In turmeric, a major spice and medicinal plant, a protocol for RNA isolation is not available. The major difficulty encountered while using other popular protocols is the low yield and quality of RNA which hampers the downstream applications like qRT-PCR, cDNA synthesis and micro RNA isolation. Commercial kits though available are costly and were found to be unsuccessful in case of rhizomes and root tissues that are rich in polyphenols, polysaccharides and alkaloids. It was thus felt that a quick, handy and cheap protocol of total RNA isolation from different tissues of turmeric was required for day to day working in our lab. The new protocol utilizes SDS based extraction buffer including β-mercaptoethanol and PVP with sequential acid phenol:chloroform extraction to remove polyphenols and proteins, followed by the purification with sodium acetate to eliminate polysaccharides. The protocol is simple and can be completed in less than 3 h. The RNA yield from rhizome was higher by more than fivefold with both A_260/280 and A_260/230 ratio in the range of 1.8–2.0. The protocol worked well with leaf, rhizome, pseudostem and root tissues with RIN >7.0 and the isolated RNA could be successfully used for cDNA synthesis, RT-PCR, qRT-PCR and small RNA isolation including microRNA.     

Rapid High Quality RNA Preparation from Pine Seedlings

Conventional RNA extraction methods have been shown to produce poor quality RNA when applied to pine and other gymnosperms. We present a protocol for extracting highly pure RNA from pine. Modifications to conventional procedures include: 1) the use of seedlings, 2) the use of phenol and PVP to rapidly remove DNA, proteins and pigments, and 3) the use of salt precipitation to remove other contaminating compounds. The procedure can be completed in less than eight hours. Yield and purity were monitored by denaturing gel electrophoresis and by UV absorbance (A₂₆₀ /A₂₈₀ and A₂₆₀/A₂₃₀). These ratios were over 2, indicating an absence of contaminating metabolites. Additionally, a new absorbance ratio (A₂₆₀/A₂₁₀) was introduced to monitor the RNA purity in each step (it indicates the ratio of covalent links in the solution belonging to RNA). The yield was around 300 µg total RNA per gram of tissue of Pinus sylvestris and over 400 µg of total RNA per gram of P. pinaster tissue, which is a high recovery (more than 63%) for gymnosperms. The RNA was of sufficient quality for use in a RT-PCR reaction that amplified 1 kb of the pine GS gene. This protocol has been applied with success to other woody plants like Populus species.Abbreviations: DEPC, diethyl pyrocarbonate; AMV, avian myeloblastosis virus; FW, fresh weight.     

Improved RNA isolation from Laminaria japonica Aresch (Laminariaceae, Phaeophyta)

RNA isolation is difficult in some plants and algae because phenolics, polysaccharides, or other compounds can bind or co-precipitate with RNA, and because the success of RNA isolation can be strain-specific and species-specific. To create an improved RNA isolation protocol for Laminaria japonica Aresch (Laminariaceae, Phaeophyta), four methods for extracting RNA were tested. A cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol was developed that clearly showed 28S and 18S ribosomal RNA bands and produced RNA with high yield (68 μg g⁻¹ fresh weight) and high quality (A _260/280 ratio 1.96 ± 0.05). The isolated RNA was intact, and RT-PCR analysis confirmed that further molecular application is feasible.     

A fast, simple, and reliable high-yielding method for DNA extraction from different plant species

Genetic studies and pathogen detection in plants using molecular methods require the isolation of DNA from a large number of samples in a short time span. A rapid and versatile protocol for extracting high-quality DNA from different plant species is described. This method yields from 1 to 2 mg of DNA per gram of tissue. The absorbance ratios (A₂₆₀/A₂₈₀) obtained ranged from 1.6 to 2.0. A minimal presence of contaminating metabolites (as polymerase chain reaction [PCR] inhibitors) in samples and a considerable savings in reagents are characteristics of this protocol, as well as the low cost of the analysis per sample. The quality of the DNA was suitable for PCR amplification.     

RNA-free DNA Extraction Protocol from PinusTissues for Molecular Biology or HPCE/HPLC Analyses

A simple method for extracting DNA from various in vitro or ex vitro Pinus tissues is described. Good yield and high quality RNA-free DNA ready to use in molecular biology assays or analytical analysis was obtained. DNA quality was measured analyzing absorption spectra between 200 and 300 nm, A_260/280 ratio and HPCE. This protocol was tested with no modification in a wide range of Pinus tissues from recalcitrant in vitro callus to mature field scions improving the results obtained with previous protocols. As the protocol is simple, almost universal and inexpensive it may be used for routine isolation from Pinus tissues. An isolation troubleshooting table was included to facilitate the setting up of reported protocol to other plant species.     

A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites

A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides. The method is based on the Nucleon PhytoPure^™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same time. The yield ranged from 240 μg up to 3 mg per gram of tissue with an average purity measured as A_260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried out with the RiboGreen^™ reagent and of PCR products with the PicoGreen^™ reagent.     

Isolation of RNA of high quality and yield from <Emphasis Type="BoldItalic">Ginkgo biloba</Emphasis> leaves

An improved protocol was developed to isolate total RNA in good yield and integrity from Ginkgo biloba leaves containing high levels of flavonoid glycosides, terpene lactones, carbohydrates and polyphenolic secondary metabolites. Polyvinylpolypyrrolidone at 2% and β-mercaptoethanol at 4% were added to the standard CTAB extraction buffer and, after chloroform and phenol extraction, the pellet obtained by ethanol/acetate precipitation was washed and a second phenol/chloroform extraction was introduced to remove co-precipitated polysaccharides. Both A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ absorbancy ratios of isolated RNA were around 2 and the yield was about 0.4 mg g^--1 fresh weight. At least seven distinct rRNA bands were detected by denaturing gel electrophoresis. Sharp hybridization signals were obtained from Northern blots with both nuclear and plastid gene probes. Two gene fragments: nuclear-encoded cab and chloroplast encoded rbcL were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total RNA isolated by this protocol is of sufficient quality for subsequent molecular applications.     

Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity

Due to their efficiency in the hydrolysis of the collagen triple helix, Clostridium histolyticum collagenases are used for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A Förster resonance energy transfer triple-helical peptide substrate (fTHP) has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence: Gly-mep-Flp-(Gly-Pro-Hyp)₄-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)₄-NH₂] had a melting temperature (T_m) of 36.2 °C and was hydrolyzed efficiently by bacterial collagenase (k_cat/K_M = 25,000 s⁻¹ M⁻¹) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity toward triple helices and, thus, potential application for evaluating the efficiency of cell isolation by collagenases.     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

An improved method for high-quality RNA isolation from needles of adult maritime pine trees

When conventional RNA isolation methods optimized for pine seedlings are applied to needles of adult pine trees, poor-quality RNA results. Here we describe a modified procedure to isolate high-quality RNA from needles of 30-year-old maritime pines, exhibiting high levels of phenolics, polysaccharides, and RNases. Major changes are the inclusion of proteinase K in the extraction medium followed by incubation at 42°C. Integrity and purity were evaluated by using denaturing gel electrophoresis and spectrophotometry (A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀). The total RNA could be successfully used for poly(A)⁺-RNA isolation and cDNA library construction.     

Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays

The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol–chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol–chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.     

Characterization of wheat root ribosomes isolated by Mg2+ precipitation

The Mg²⁺ precipitation method has been adapted for isolation of ribosomes from roots of wheat. The ribosomes prepared by this procedure show A₂₆₀/A₂₈₀ = 1.6 and A₂₆₀/A₂₃₅ = 1.3 and contain 44d% RNA and 56% ribosomal proteins. There are no detectable differences in the ribosomal protein complement and accessibility of the ribosomal proteins to phosphorylation between ribosomes isolated by this procedure and those prepared by classical ultracentrifugation methods. The ribosomes are active in a poly-U directed cell-free system for protein synthesis.     

Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 g g^–1 fresh weight) with high quality (A_260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies.