Fine-needle aspiration technique under endoscopic ultrasound guidance:A technical approach for RNA profiling of pancreatic neoplasms

BACKGROUND Early diagnosis of pancreatic ductal adenocarcinoma(PDAC)has been a longstanding challenge.The prognosis of patients with PDAC depends on the stage at diagnosis.It is necessary to identify biomarkers for the detection and differentiation of pancreatic tumors and optimize PDAC sample preparation procedures for DNA and RNA analysis.Most molecular studies are done using paraffin-embedded blocks;however,the integrity of DNA and RNA is often compromised in this format.Moreover,RNA isolated from human pancreatic tissue samples is generally of low quality,in part,because of the high concentration of endogenous pancreatic RNAse activity present.AIM To assess the potential of endoscopic ultrasound-guided fine-needle aspiration(EUS-FNA)to obtain specimens from pancreatic neoplasms for subsequent RNA molecular profiling,including next-generation sequencing(NGS).METHODS Thirty-four EUS-FNA samples were included in this study:PDAC(n=15),chronic pancreatitis(n=5),pancreatic cysts(n=14),mucinous cysts(mucinous cystic neoplasia/intraductal papillary mucinous neoplasia)n=7,serous cystic neoplasms n=5,and pseudocysts n=2.Cyst material consisted of cyst fluid and cyst wall samples obtained by through-the-needle biopsy(TTNB).Samples were stored at -80℃ until analysis.RNA purity(A260/230,A260/280 ratios),concentration,and integrity(RIN)were assessed.Real-time polymerase chain reaction was conducted on all samples,and small RNA libraries were prepared from solid mass samples.RESULTS RNA was successfully extracted from 29/34(85%)EUS-FNA samples:100% pancreatic adenocarcinoma samples,100% chronic pancreatitis samples,70% pancreatic fluid cyst samples,and 50%TTNB samples.The relative expression of GAPDH and HPRT were obtained for all successfully extracted RNA samples(n=29)including lowquality RNA specimens.Low concentration and nonoptimal RIN values(no less than 3)of RNA extracted from EUS-FNA samples did not prevent NGS library preparation.The suitability of cyst fluid samples for RNA profiling varied.The quality of RNA extracted from mucinous cyst fluid had a median RIN of 7.7(5.0-8.2),which was compatible with that from solid neoplasms[6.2(0-7.8)],whereas the quality of the RNA extracted from all fluids of serous cystic neoplasms and TTNB samples had a RIN of 0.CONCLUSION The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions,including low-quality RNA specimens.

Comparison of RNA Extraction Methods of Sugarcane Stem

[Objective] The study was to explore the most effective and feasible method for sugarcane stalks RNA extraction.[Method] SDS extraction method,kit extraction method and GHCL extraction method were used to extract the sugarcane RNA in stalks,and the quality of the extracted RNA was compared.[Result] RNA extracted by kit extraction method had a high-yield,the bands were clear and RNA had a good integrity,there was no significant degradation of RNA,and the OD260 nm/OD280 nm value was closed to 2.0.[Conclusion] Kit extraction method was the effectively method to extract sugarcane RNA,and this study had provided a theoretical basis for the molecular biology study of sugarcane.

RNA Extraction from Herba Violae Roots with Low-temperature Sectioning Method

[ Objective ] This study aimed to investigate the optimal method for extracting RNA from roots of medicinal plant herba violae by comparing the effects of liquid nitrogen grinding method and low-temperature sectioning method on RNA extraction. [ Method] Roots of herba violae were respectively crushed by using liquid nitrogen grinding method and low-temperature sectioning method to extract RNA. The extraction effects of these two methods were compared based on detec- tion of RNA concentration, purity and integrity and amplification of GAPDH gene by RT-PCR. [Result] The concentration of RNA extracted by liquid nitrogen grinding method and low-temperature sectioning method was 1.21 and 3.57 p^g/~, respectively. Both RNA extracted by these two methods showed two distinct bands after agarose gel electrophoresis. The ratio of brightness of the 28S rRNA to the 18S rRNA bands was greater than 1. PCR amplification showed that the length of GAPDH gene was about 230 bp, which was consistent with the expected result. [ Conclusion ] The experimental results indicated that using low-tempera ture sectioning method to crush the roots of herba violae can meet the needs of most molecular biological experiments including gene cloning and expression analysis, which is an effective and simple method for extracting RNA from plant roots.

An improved method for RNA extraction from urediniospores of and wheat leaves infected by an obligate fungal pathogen, Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation of P. striiformis f. sp. tritici winter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR （qRT-PCR）. A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefficient. Thus, we modified three previously reported RNA extraction methods with common and low-cost reagents （LiCI, SDS and NaCI） to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCI method was proven to be the best for extracting RNAfrom urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified LiCI and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCI method has the following advantages： 1） Complete transfer of urediniospores of P. striiformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2） the use of low-cost NaCI to replace more expensive Trizol can reduce the cost; 3） the yield and quality of RNA can be increased; 4） the improved method is more suitable for a large number and high quantity of samples from fields. Using the improved NaCI method, the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared from the extraction kit. Approximately, 10.11 IJg total RNA of high quality was obtained from 100 mg of infected leaves, which was 8.8, 6.5, 3.4 and 2.1 folds of the amounts obtained from the previous LiCI, SDS, NaCI and traditional Trizol methods, respectively. The method could be used to study the overwintering rates of R striiformis f. sp. tritici over a large region of wheat production for predicting epidemic levels by determining pathogen survival levels after winter. The method can alsobe used in any studies which need a large number of high quality RNA samples.

Comparative Study on the Extraction of Total RNA from Different Tissue Parts of Cone Snail(Conus geographus)

[Objectives] This study was conducted to compare the quality of total RNA extracted from different tissues of cone snail( Conus geographus).[Methods] The total RNA of four different tissues of cone snail,venom gland,venom tube,salivary gland and tooth gland was extracted by the Trizol method. The total RNA of cone snail was detected by agarose gel electrophoresis,NanoDrop^(TM) 2000 spectrophotometer and Aligent 2100 biological analyzer. [Results]The total RNA extracted from different tissues of cone snail showed clear band,and thus had similar concentrations and purity,and the highest yield was obtained from venom tube. [Conclusions] The total RNA extracted from different tissue parts of cone snail could meet the basic requirements of molecular biology,and its venom tube was the best tissue part for extracting total RNA,which lays a foundation for molecular biology research such as high-throughput transcriptome sequencing of cone snail.

A Comparative Study on Extraction Methods of Total RNA from Leaves of Acer truncatum ‘Luhong No.1’

Leaves of Acer truncatum ＇ Luhong No. 1 ＇ contain large amounts of polysaccharides and polyphenols, which seriously affect extraction yield and quality of total RNA. In order to explore the appropriate total RNA extraction method, total RNA was extracted from leaves of A. truncatum ＇ Luhong No. 1 ＇ with three methods, including kit method, Trizol method and modified CTAB method. The results showed that ODE6o/OD2so and OD^o/OD2ao ratios of total RNA extracted from leaves of A. truncatum ＇ Luhong No. 1 ＇ with kit method were higher than 1.8, with a general yield and certain level of DNA contamination ; OD^o/OD~ and OD^o/OD^o ratios of total RNA extracted with Trizol method were about 1.5, with the lowest yield; OD260/OD280 and OD260/OD230 ratios of total RNA extracted with modified CTAB method were about 2.0, with the highest yield and distinct eleetrophoresis patterns. The results demonstrated that total RNA extracted with modified CTAB method exhibited high yield and purity, which could meet the demands of subsequent molecular biology research. Thus, modified CTAB method is the appro- oriate method for extracting total RNA from leaves of A. truncatum ＇ Luhong No. 1 ＇

An Effective Method for Extracting Total RNA from Young Embryo of Seedless Litchi

A total RNA extraction method for young embryo of seedless litchi was introduced. CTAB, Phenol （saturated with water）, chloroform, Guanidine isothioeyanate were used as main extraction reagents. Polyphenolie compounds were removed effectively by added PVP into the extraction buffer solution. RNA was purified intensively by phenol, chloroform extraction, and ethanol deposition after deposited by LiCl. Both the results of formaldehyde denatured agarose gel eleetrophoresis and ultraviolet spectrophotometer analysis showed high integrity and purity of RNA. So the quality of extracted RNA could meet the demand of most molecular biology experiments that require higher quality RNA.

Comparison of methods for extracting high-throughput sequencing RNA from Korean pine seeds

Korean pine （Pinus koraiensis Sieb. et Zucc.） is an ecologically and economically important tree species in East Asia. Molecular studies of seed development of this species are limited due to the lack of effective RNA extraction protocols. This study aimed to obtain an effec- tive method to extract high-quality RNA from Korean pine seeds. The TRIzol kit and CTAB methods were used to extract the total RNA from Korean pine seeds at different developmental stages. The bands of RNA extracted by CTAB were not clear, whereas the bands of RNA extracted by the TRIzol kit were brighter and clearer, indicating higher quality and integrity of the RNA products extracted by the TRIzol kit. The 28S rRNA band was approximately 1.5- to 2-fold brighter than the 18S rRNA band on the agarose gel electrophoresis. The absorbance value A 260/280 was 1.8-2.0, and the absorbance value A 260/230 was 〉1.9. The Bioanalyzer RNA integrity results showed that the RNA integrity number of the RNA extracted using the TR/zol kit was acceptable for high-throughputsequencing. Therefore, the total RNA extracted using the TRIzol kit method can be used for high-throughput sequencing and other molecular biology experiments.

A Non-toxic and Efficient Method for Extracting DNA and RNA from Peanut

[Objectives]To establish a non-toxic and efficient method for extracting DNA and total RNA from peanuts and laying a solid foundation for the molecular biology study of peanuts.[Methods]Based on the principle and method of purifying nucleic acids by silica gel adsorption at high salt and low pH condition,a non-toxic and efficient method to extract peanut DNA and total RNA using cetyltrimethyl ammonium bromide(CTAB)extraction solution was designed.The quality and purity of nucleic acids were detected by agarose gel electrophoresis and nucleic acids protein analyzer,respectively.The quality of DNA was further verified by enzyme digestion and PCR amplification using molecular marker techniques.The quality of total RNA was further verified by reverse transcription(RT)-PCR of actin gene and cDNA-SCoT gene differential display technique.[Results]The agarose gel electrophoresis test showed that the peanut DNA extracted by a low-toxic and effective method is free of contamination and degradation.Through the detection by the nucleic acid protein analyzer,the DNA concentration,yield,A260/A280 and A260/A230 of 5 peanut varieties were 419.6-498.2 ng/μL,20.98-24.91μg/g,1.89-1.96 and 2.03-2.28,respectively.The DNA was of high quality and can be completely digested by EcoRI restriction enzymes,and also can be used for SCoT and SRAP molecular marker technology analysis.The RNA extracted from different tissues of peanuts showed no visible DNA bands by non-denaturing agarose gel electrophoresis.The separated 28S bands were brighter than 18S.The ratio of A260/A280 and A260/A230 showed that the RNA quality was good and can be used for reverse transcription,RT-PCR of actin gene and amplification of cDNA-SCoT gene differential display technique.[Conclusions]This experiment established a low-toxic and effective method for extracting DNA and total RNA from peanuts.Compared with traditional methods,this method is more time-saving and cheaper than commercial kits.The most important point is that this method does not use toxic reagents such as phenol,chloroform and isopropanol.Thus,it is expected to be widely applied in molecular biology research.

Cloning of TRP Gene of Bombyx mori

[Objective]The aim was to clone Bombyx mori TRP gene.[Method]The total RNA of Bombyx mori was extracted during its pupal period with Trizol method,and then the TRP gene was cloned by RT-PCR.[Result]The full-length cDNA of TRP gene was successfully obtained,and the ORF fragment（858 bp） of TRP gene was cloned by PCR.[Conclusion]TRP gene of Bombyx mori was cloned for the first time,which could provide solid basis for the study on its function.

Improved Nucleic Acid Spot Hybridization Technique for Detection of Potato Spindle Tuber Viroid(PSTVd)

Potato spindle tuber viroid(PSTVd)disease is one of the major diseases that threatens potato production.Therefore,an advanced,rapid and sensitive detection technology is needed to detect the disease for better control.In order to establish an easier nucleic acid spot hybridization(NASH)method,some studies were tried as the followings:(1)the pre-hybridization step of nucleic acid spot hybridization(NASH)was omitted compared with ordinary way;(2)RNA extraction(phenol extraction and Ames buffer extraction)methods were compared;(3)fixed RNA by UV lamp and oven compared with UV cross-linker;(4)hybridized the RNA in shaking incubator and so on.The results showed that RNA extracted by Ames buffer was more effective than by the phenol extraction method.Besides,the result of hybridization without pre-hybridization step was better than that with 1.5 h of pre-hybridization.The more important discovery was that the shaking incubator could replace the hybridization oven and the ordinary UV lamp could replace the UV cross-linker.After a long term repeated research and testing,a new hybridization system that could rapidly detect the PSTVd by improved NASH technique merely using common instruments and equipment was established.