OBJECTIVETo establish a method for isolation of the total RNA from Fagopyrum cymosum callus.

METHODThe improved method combining that of CTAB extraction with the LiCl precipitation was used to isolate the total RNA from the four F. cymosum callus. The quality of the RNA was detected by UV spectrophotometric analysis, 0.8% non-denaturing agarose gel electrophoresis and RNA reverse transcription.

RESULTThe bands of 28S and 18S could be seen clearly by agarose gel electrophoresis, and the value of A260/A280 was between 1.9 and 2.0. The cDNA which was reverse-transcribed by the total RNA showed a wide length rage of 500 bp-5 kb.

CONCLUSIONThe RNA extracted by this method meets the requirement of reverse transcription-polymerase chain reaction (RT-PCR), construction of cDNA libraries, et al. This improved method can be used to isolate the total RNA from F. cymosum callus with the advantage of simpleness, efficiency and low cost.

Fagopyrum ; genetics ; growth & development ; RNA, Plant ; analysis ; isolation & purification

A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.
Animals ; DNA, Complementary ; chemistry ; isolation & purification ; Gene Library ; RNA ; isolation & purification ; Sea Anemones ; genetics

OBJECTIVETo explore a convenient and effective method for norovirus nucleic acid extraction from oysters suitable for long-term viral surveillance.

METHODSTwo methods, namely method A (glycine washing and polyethylene glycol precipitation of the virus followed by silica gel centrifugal column) and method B (protease K digestion followed by application of paramagnetic silicon) were compared for their performance in norovirus nucleic acid extraction from oysters. Real-time RT-PCR was used to detect norovirus in naturally infected oysters and in oysters with induced infection.

RESULTSThe two methods yielded comparable positive detection rates for the samples, but the recovery rate of the virus was higher with method B than with method A.

CONCLUSIONMethod B is a more convenient and rapid method for norovirus nucleic acid extraction from oysters and suitable for long-term surveillance of norovirus.

Animals ; Centrifugation ; methods ; Norovirus ; genetics ; isolation & purification ; Ostreidae ; virology ; RNA, Viral ; isolation & purification

BACKGROUNDMitochondrial DNA mutations have been found in sensorineural deafness. The aim of this study was to compare three methods for extraction of nucleic acid from membranate inner ear tissue of rats.

METHODSAlkaline denaturation, a conventional phenol-chloroform method and Trizol reagent were respectively used to extract the slight nucleic acid from membranate inner ear tissue of rats. We assessed the amount and quality of nucleic acid using a UV-spectrometer and polymerase chain reaction (PCR).

RESULTSThe yield and purity (OD260/OD280) of DNA from inner ear tissue using the phenol-chloroform method was the highest of the three methods. Mitochondrial DNA (mtDNA) fragment can be amplified by PCR from nucleic acid prepared by all methods, while no nuclear DNA (nDNA) fragment can be amplified by method of alkaline denaturation. Both nuclear and mitochondrial genes could be amplified by reverse transcriptional PCR from the RNA prepared by Trizol reagent.

CONCLUSIONAdequate amount and high-quality of mtDNA, nDNA and RNA were obtained from unilateral membranate inner ear tissue of rats. Method of alkaline denaturation could be chosen when mtDNA without nDNA was needed, while phenol-chloroform method was suitable for extracting total DNA (including nDNA and mtDNA); method with Trizol reagent was suitable for extracting total RNA and total DNA.

Animals ; DNA ; isolation & purification ; DNA, Mitochondrial ; isolation & purification ; Ear, Inner ; chemistry ; Polymerase Chain Reaction ; RNA ; isolation & purification ; Rats ; Rats, Wistar

Chikungunya virus ; genetics ; isolation & purification ; China ; Genome, Viral ; RNA, Viral ; genetics ; Sequence Analysis, RNA

To investigate the method of RNA isolation from human embryonic tissues and the factors influencing the quality of RNA, the RNA from human embryonic tissues obtained with drug-induced labor or non-drug induced labor were isolated by using grind with liquid nitrogen or homogenizer without liquid nitrogen. The results showed that the positive rates of RNA integrity in grind with liquid nitrogen group and in homogenizer without liquid nitrogen group were 68.42% and 29.79% respectively, and there was significant difference between these two groups; however, there was no statistic difference in positive rate of RNA integrity, OD(260)/OD(280) ratio and beta-actin gene expression level between the drug-induced labor group and non-drug induced labor group. It is concluded that pulverize of tissue in liquid nitrogen remains the integrity of RNA isolated and may be applied for RNA isolation from human embryonic tissues. The quality of RNA is not affected by different methods of induction of maternal labor.
Embryo, Mammalian ; metabolism ; Freezing ; Humans ; Nitrogen ; pharmacology ; RNA ; isolation & purification ; RNA Stability ; drug effects

OBJECTIVETo develop a reliable, rapid assay for detecting pathogenic aspergillus species in fungal sinusitis.

METHODSThirty-seven formalin-fixed and paraffin-embedded surgical tissue specimens from patients with fungal sinusitis were used in the present study. The aspergillus specific oligonucleotide probe was designed, commercially synthesized, and digoxigenin-labeled. Twenty-three-base oligonucleotides was selected that was complementary to 18S ribosomal RNA sequences (18S-1 probe) for detecting medically important aspergillus species.

RESULTSIn situ hybridization for aspergillus rRNA was positive in 28 cases with the 18S-1 probe. Compared with HE (21) and methenamine-silver stain (23).

CONCLUSIONIn situ hybridization provides rapid and accurate identification for fungal organism in tissues, and may be useful if cultures are negative or have not performed.

Adult ; Aged ; Aspergillosis ; diagnosis ; Aspergillus ; genetics ; isolation & purification ; Female ; Humans ; In Situ Hybridization ; Male ; Middle Aged ; RNA, Fungal ; isolation & purification ; RNA, Ribosomal, 18S ; isolation & purification ; Sinusitis ; diagnosis ; microbiology

OBJECTIVETo explore the value of detecting bacterial 16S rRNA with 23S rRNA in the diagnosis of periprosthetic joint infection (PJI).

METHODSA prospective study was conducted among 67 patients with previous total hip arthroplasty (THA) undergoing a reoperation for infection (23 patients) or aseptic loosening (44 patients). Bacterial 16S rRNA and 23S rRNA in the interface membrane were detected by real-time PCR and their value in diagnosis of PJI was assessed.

RESULTSThe 16S rRNA and 23S rRNA showed no significant difference in their power in the diagnosis of PJI. The detection of 16S rRNA/23S rRNA showed a higher sensitivity and a greater negative predictive value in PJI diagnosis than the detection of 16S rRNA+23S rRNA (95.7% vs 52.2%, P<0.01; 97.6% vs 79.6%, P=0.01). The specificity, positive predictive value, and accuracy of the 4 diagnostic strategies were not significantly different.

CONCLUSIONSThe diagnostic power of 16S rRNA and 23S rRNA was similar in detecting PJI. Compared with the diagnostic strategy with 16S rRNA+23S rRNA, 16S rRNA/23S rRNA is more sensitive in detecting PJI.

Arthritis, Infectious ; diagnosis ; microbiology ; Arthroplasty, Replacement, Hip ; Humans ; Prospective Studies ; Prosthesis-Related Infections ; diagnosis ; microbiology ; RNA, Bacterial ; isolation & purification ; RNA, Ribosomal, 16S ; isolation & purification ; RNA, Ribosomal, 23S ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Reoperation ; Sensitivity and Specificity

Pathogens like bacteria and protozoa, which affect human and animal health worldwide, can be transmitted by vectors like ticks. To investigate the epidemiology and genetic diversity of bacteria and protozoans carried by ticks in Chengmai county of Hainan province, China, 285 adult hard ticks belonging to two species [
Anaplasmataceae/isolation & purification* ; Animals ; Chaperonin 60/genetics* ; China ; Citrate (si)-Synthase/genetics* ; Coccidia/isolation & purification* ; Coxiellaceae/isolation & purification* ; Insect Vectors/microbiology* ; Islands ; Ixodidae/microbiology* ; Phylogeny ; Piroplasmia/isolation & purification* ; RNA, Ribosomal, 16S/genetics* ; RNA, Ribosomal, 18S/genetics*

Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein-RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein (PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.
Binding Sites ; Cell Line ; Heterogeneous-Nuclear Ribonucleoproteins ; isolation & purification ; metabolism ; Humans ; Immunoprecipitation ; methods ; Polypyrimidine Tract-Binding Protein ; isolation & purification ; metabolism ; RNA ; isolation & purification ; metabolism ; RNA-Binding Proteins ; isolation & purification ; metabolism