A new strategy for introducing photoactivatable 4-thiouridine ((4S)U) into specific positions in a long RNA molecule.

We describe a new protocol, which does not require (4S)UpG, for introducing (4S)U into specific sites in a pre-mRNA substrate. A 5'-half and a full-length RNA are first synthesized by phage RNA polymerase. p(4S)Up, which is derived from (4S)UpU and can therefore be 32P-labeled, is then ligated to the 3' end of the 5'-half RNA with T4 RNA ligase. The 3' phosphate of the ligated product is removed subsequently by CIP (calf intestinal alkaline phosphatase) to produce a 3'-OH group. The 3'-half RNA with a 5' phosphate is produced by site-specific RNase H cleavage of the full-length pre-mRNA directed by a 2'-O-methyl RNA-DNA chimera. The two half RNAs are then aligned with a bridging oligonucleotide and ligated with T4 DNA ligase. Our results show that 32P-p(4S)Up ligation to the 3' end of the 5'-half RNA is comparable to 32P-pCp ligation. Also, the efficiency of the bridging oligonucleotide-mediated two-piece ligation is quite high, approximately 30-50%. This strategy has been applied to the P120 pre-mRNA containing an AT-AC intron, but should be applicable to many other RNAs.     

In situ localization of small RNAs in plants by using LNA probes

Small RNAs have crucial roles in numerous aspects of plant biology. Despite our current understanding of their biogenesis and mechanisms of action, the biological function of small RNAs, particularly miRNAs, remains largely unknown. To decipher small RNA function, knowledge about their spatiotemporal patterns of expression is essential. Here we report an in situ hybridization method for the precise localization of small RNAs in plants by using locked nucleic acid (LNA) oligonucleotide probes. This method has been adapted from protocols used to detect messenger RNAs in formaldehyde-fixed and paraffin-embedded tissue sections, but it includes essential optimizations in key prehybridization, hybridization and posthybridization steps. Most importantly, optimization of probe concentration and hybridization temperature is required for each unique LNA probe. We present the detailed protocol starting from sectioned tissues, and we include troubleshooting tips and recommended controls. This method has been used successfully in several plant species and can be completed within 2-6 d.     

Small RNA library preparation for next-generation sequencing by single ligation,extension and circularization technology

The discovery of novel small RNA classes and species has accelerated since the implementation of high-throughput sequencing technologies for the identification of small RNAs. However, as the sequence coverage increases in a cell, the expectation of finding novel small RNAs from a batch of sequencing gradually decreases. To improve the finding of novel small RNAs, an alternative small RNA library preparation method, the single ligation, extension and circularization method, has been developed which is adequate for high throughput sequencing. The procedure is faster and simpler than the more widely used procedures, and the constructed libraries are compatible with high-level multiplex analysis. The analysis of human small RNA libraries prepared by the SLEC method reported known small RNAs and novel small RNAs including 25 mirtron candidates. This study demonstrates that the method is effective in identifying known and novel small RNAs.     

A systematic, ligation-based approach to study RNA modifications

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.     

A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA

Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem–loop reverse primer-based protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem–loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs.     

Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.

The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures.     

RNA microarray for estimating relative abundance of 16S rRNA in microbial communities

We developed an RNA microarray protocol in which total RNA from a microbial community was attached to a slide glass, and rRNA was detected by fluorescently labeled oligonucleotide probes. The RNA microarray requires only 4 h for hybridization and enables double staining and estimating relative abundance of rRNA.     

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%¹. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 µl of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/µl from serum but can also be adapted for other biological tissues.     

Effect of alpha-actinin on actin structure. Actin ATPase activity

The accumulation of low molecular weight RNAs in Escherichia coli cells following amino acid or energy source starvation was examined using two-dimensional polyacrylamide gel electrophoresis. 32P-labeled small RNA prepared from serine- or isoleucine-starved stringent strain (relA+) cells was shown to display gel patterns that were grossly different from that of unstarved cells. It appears that the deprivation of serine or isoleucine has little or no inhibitory effect on the accumulation of transfer RNA cognate to the deprived amino acid. This is demonstrated by a relative increase in the concentrations of small RNAs that can be charged with serine or isoleucine following starvation of these amino acids. However, small RNAs labeled during starvation of phenylalanine or energy source showed gel patterns similar to that of control cells. This suggested a heterogenous response in the accumulation of some low molecular weight RNAs, presumably transfer RNAs, following starvation of different amino acids.     

植物小RNA荧光原位杂交实验方法

小RNA是对植物生长发育十分重要的一类小分子核苷酸,在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能,而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小RNA进行定性或半定量分析的技术,目前该技术已经在动物体内被广泛应用,而在植物体内的应用还比较少。该文...     

Sea urchin small nuclear RNA genes are organized in distinct tandemly repeating units.

The genes coding for the two major small nuclear RNAs in the sea urchin are organized in independent tandem repeating units. The small nuclear RNAs, N1 and N2 were purified from gastrula embryos of Lytechinus variegatus. These RNAs are analogous to the U series of RNA in mammalian cells as judged by their identical 5' termini and the sequence homology of the N1 urchin RNA and U1 mouse RNA. These RNAs were polyadenylated with E. Coli adenylate transferase. A 32PO4 labeled copy of each RNA was made with RNA-dependent DNA polymerase. This copy was used to probe the gene organization of these RNAs by hybridizing to restriction enzyme digests of sperm DNA. Each of these RNAs is coded in a tandemly repeated cluster (at least 30 kb) with a repeat length of 1100-1400 bases. The N1 and N2 clusters are distinct. The N1 repeat has been cloned and the repeating organization confirmed with the cloned gene.     

Isolation and cloning of microRNAs from recalcitrant plant tissues with small amounts of total RNA: a step-by step approach

MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21–24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.     

Recent developments in methods for RNA sequencing using in vitro 32P-labeling

A variety of approaches that utilize in vitro 32P-labeling of RNA and of oligonucleotides in the sequence analysis of RNAs are described. These include 1) methods for 5'- and 3'- end labeling of RNAs; 2) end labeling and sequencing of oligonucleotides present in complete T1 RNase or pancreatic RNase digests of RNA; 3) use of random endonucleases, such as nuclease P1, for terminal sequence analysis of end labeled RNAs; and 4) use of base specific enzymes or chemical reagents in the sequence analysis of end-labeled RNAs. Also described is an approach to RNA sequencing, applied so far to tRNAs, which is based on partial and random alkaline cleavage of an RNA to generate a series of overlapping oligonucleotide fragments, all containing the original 3'-end of the RNA. Analysis of the 5'- end group of each of these oligonucleotides (following 5'-end labeling with 32P) provides the sequence of most of the tRNA. The above methods have been used to derive the sequences of several tRNAs, the ribosomal 5S and 5 x 8S RNAs, a viroid RNA, and large segments of both prokaryotic and eukaryotic ribosomal and messenger RNAs.     

小RNAs与其靶标的相互调控

小RNAs 是生命活动非常重要且广泛存在的调节因子,可以调控基因表达和基因组稳定性. 然而最近的研究发现,小RNAs与它们的靶标间的调控是相互的调控(reciprocal regulation),因为它们的靶标反过来也可以调控小RNAs. 也就是说,它们的靶标可以通过自身与小RNA的互补程度或自身丰度水平,引发小RNAs无需模板地在其3′ 端添加核苷酸,导致小RNAs降解. 另外,病毒也可以通过这种方式调控宿主基因的表达. 这种现象的发现挑战了以前对小RNAs作用过程的理解,这不仅可以解释以前一些关于小RNAs所不能解释的问题,而且对于转基因技术、反义核酸技术和RNA干扰技术都有重要的启示作用. 本文综述了这种靶标引发小RNA修饰并降解现象的研究进展和应用前景.