制样方法对菊花花蕾激光显微切割样品RNA提取质量的影响

[目的]研究不同固定剂及切片制样方式对菊花花蕾激光显微切割样品RNA提取质量的影响。[方法]以丙酮和乙醇冰醋酸2种试剂固定菊花花蕾,采用石蜡和冰冻切片2种方式制作样本,显微切割并收集最外两轮小花原基,提取RNA后比较提取质量。[结果]石蜡和经梯度蔗糖保护的冰冻切片均能很好地保存花蕾组织形态,就石蜡切片而言,乙醇冰醋酸固定组织切割样品的RNA降解明显低于丙酮固定样品;而2种固定剂的冰冻切片切割样品RNA提取质量无明显差异。与石蜡切片相比,冰冻切片显微切割样品RNA质量更高。切割时收集管盖加入RNA提取缓冲液可明显缓解RNA降解。显微切割的不同品种菊花花原基与花发育有关的基因表达水平存在差异,表明切割获得的材料可用于下一步研究。[结论]植物组织经乙醇冰醋酸固定、梯度蔗糖保护后冰冻切片进行显微切割效果较佳,可富集特异组织或细胞并从中获得较高质量的RNA。

改良CTAB法提取不同作物总RNA技术研究

为提高操作效率,本研究开发出一种简洁有效的作物总RNA提取方法。研究以油菜、棉花、花生、小麦、马铃薯、玉米、大豆、西葫芦等作物的不同组织为试材,通过CTAB法、改良CTAB法及TanSzol Reagent试剂盒法提取总RNA,检测结果发现,经过改良后的CTAB法提取的总RNA质量较高。在油菜花瓣、小麦、花生、西葫芦叶片中,该方法获得了较高的总RNA得率,分别为24.5、23.7、26.4、17.35μg/mL;OD260/280和OD260/230值分别为1.90、1.73、1.72、1.80和2.10、1.50、2.01、1.70。综合比较以上3种方法,改良后的CTAB法适用于油菜、小麦、花生、西葫芦等作物的总RNA提取,为后续分子生物学检测奠定了基础。

四种类型竹种竹叶总RNA提取方法的比较研究

为探索适合不同类型竹种竹叶总RNA的提取方法,本研究选取合轴丛生型竹种勃氏甜龙竹、合轴散生型竹种云南箭竹、单轴散生型竹种毛竹、复轴混生型竹种巴山木竹为代表,提取其心叶的总RNA,通过琼脂糖凝胶电泳检测RNA的完整性,利用超微量紫外分光系统检测RNA的纯度和浓度,比较自配TRIzol试剂法、商品化TRIzon试剂法、RNA提取试剂盒法、改良十六烷基三甲基溴化铵(CTAB)法四种方法提取的竹叶总RNA的质量、纯度和提取成本的差异。结果表明,自配TRIzol试剂法可以提取出质量好、浓度高的竹叶总RNA,且提取成本较低;商品化TRIzon试剂法提取的总RNA质量及浓度仅次于自配TRIzol试剂法,提取成本稍高;而RNA提取试剂盒法和改良CTAB法无法提取出高浓度的竹叶总RNA。本研究结果为竹类植物更深入的分子生物学研究提供了一定的基础,并为其他竹类植物总RNA的提取提供了参考依据。

从牦牛乳中提取RNA用于乳腺特异蛋白编码基因的扩增

为建立从乳中提取RNA并扩增基因的方法,从4头麦洼牦牛混合乳中用超速离心法初步分离外泌体后,用TRIzol法提取其中的总RNA,经反转录后用PCR技术扩增乳腺上皮细胞3个特异基因的编码区序列并直接测序.结果显示,乳中提取的RNA质量较好,可以扩出κ-酪蛋白基因和β-乳球蛋白基因的完整编码区,长度分别为705 bp和636 bp,产物可直接测序,但未能扩增出乳铁蛋白基因.此外,本试验初步表明,从牦牛乳中提取RNA用于PCR扩增基因时,其循环数应在35循环到45循环之间.本研究建立了利用牦牛乳中外泌体提取RNA的新方法,提取的RNA可用于扩增基因和测序.

基于LiCl法优化酿酒葡萄叶片的RNA提取

为获得纯度高、质量好的酿酒葡萄植株叶片RNA以用于荧光实时定量分析,为后续以酿酒葡萄叶片RNA为材料研究相关转录水平的调控提供技术保障,以经典酿酒葡萄赤霞珠叶片为试验材料,比较分析了重蒸酚法、Trizol法、改良的十六烷基三甲基溴化铵(CTAB)法和LiCl法(基于改良的SDS法)4种方法对酿酒葡萄叶片RNA提取的影响。结果表明,重蒸酚法提取RNA浓度较低,完整性较差,存在部分降解;Trizol法提取的RNA则有部分DNA污染;改良CTAB法提取浓度较高,但蛋白和DNA污染严重;LiCl法提取结果显示,28S的条带亮度是18S的2倍,条带无拖尾,完整性较好,存在DNA污染和少量的蛋白污染。进一步在LiCl法基础上利用醋酸钠、DNAase消化、苯酚抽提法进一步优化,并采用荧光实时定量验证,结果表明,醋酸钠的加入RNA质量无明显改善;然而,经氯仿和水饱和酚(体积比1∶1)抽提2次,氯仿抽提1次,氯仿和水饱和酚(体积比1∶1)抽提1次,并经DNAase进行消化,最终提取出质量浓度为173.611μg/mL、A_(260/280)为1.803纯度高、完整性较好且无蛋白和DNA污染的葡萄叶片总RNA;经实时荧光定量PCR进一步验证,该方法提取的RNA经反转录后获得较好的扩增曲线以及融解曲线,并能够对相关基因进行定量分析。

植物RNA作为载体RNA在病毒核酸提取过程中的保护效果研究

目的探究植物RNA作为载体RNA在病毒核酸提取过程中对目标核酸的保护效果。方法分别提取绿萝、水稻、竹子3种常见植物中的RNA作为载体RNA,同时以商业试剂Takara载体RNA为对照,提取甲型流感病毒(Flu A)、乙型流感病毒(Flu B)、呼吸道合胞病毒A型(RSV A)核酸,并利用实时荧光定量反转录聚合酶链反应(RT-qPCR)定量分析提取得到的病毒核酸浓度,以评估植物RNA在核酸提取中对病毒核酸的保护效果。结果经琼脂糖凝胶电泳检测,所提取植物RNA的28S rRNA、18S rRNA条带清晰明亮,无弥散现象,所得RNA具有良好的完整性。且经验证,植物RNA对病毒核酸扩增无干扰。裂解液中分别加入0、1000、3000、5000、7000、9000 ng绿萝RNA进行Flu A RNA提取时,提取物经RT-qPCR扩增后的Ct值分别为35.06±0.14、33.01±0.42、32.45±0.33、31.95±0.34、31.74±0.28、31.92±0.24,使用Takara载体RNA提取核酸的Ct值为31.96±0.34。Ct值随着绿萝RNA添加量增加而降低,当添加量≥5000 ng时,Ct值接近使用Takara载体RNA的提取组,继续提高绿萝RNA用量,Ct值趋于稳定,且与使用TaKaRa载体RNA的Ct值比较,差异无统计学意义(P>0.05)。添加5000 ng绿萝RNA与添加0 ng绿萝RNA相比,Ct值相差3.11,根据浓度差等于2ΔCt计算可知添加5000 ng绿萝RNA提取得到的Flu A核酸浓度比不添加载体RNA高了8.63倍。进一步研究发现,添加绿萝RNA、水稻RNA、竹子RNA及Takara载体RNA,提取得到的Flu A、Flu B和RSV A核酸经RT-qPCR扩增后得到的Ct值与不添加载体RNA比较,差异均有统计学意义(P<0.05)。绿萝RNA作为载体RNA在提取Flu A、Flu B、RSV A混合病毒核酸时,对比无载体RNA的对照组,分别降低了2.61、2.90、1.45个Ct值,即添加了绿萝RNA后提取Flu A、FluB、RSV A所得的核酸浓度分别提高了6.11、7.46、2.73倍。且添加了水稻RNA和竹子RNA提取混合病毒核酸所得的核酸浓度也分别至少提高了2.63倍和2.60倍。结论在病毒核酸提取过程中植物RNA具有保护病毒核酸的作用,可以提高提取得到的目的病毒RNA浓度,可用作病毒核酸提取时的载体RNA,为核酸提取中的载体RNA选择提供更多来源。

基于SYBR Green的实时qPCR法定量检测动物脂肪中的RNA

建立一种从食用动物脂肪中提取RNA,并采用基于SYBR Green的实时荧光定量PCR(Quantitative Real-Time PCR,RT-qPCR)测定不同动物脂肪中RNA表达水平的方法。结果表明,改进后的TRIzol^((R))提取方法能够从动物脂肪中有效提取出200~500 ng·μL^(-1)的高纯度RNA。经RT-qPCR反应后所有提取的RNA样本均展现出了优异的扩增性能。本研究开发的TRIzol^((R))提取法结合RT-qPCR的新方法,可为后续动物脂肪的鉴别和追溯提供数据支撑。

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.

NucliSens miniMag与QIAamp系统检测血清丙型肝炎病毒RNA效能比较

目的比较研究NucliSens miniMag与QIAamp系统检测血清丙型肝炎病毒(HCV)RNA的效能。方法2021年6月~2023年6月我院收集的103例抗-HCV阳性血清标本,分别采用NucliSens miniMag系统(方法A)和QIAamp Viral RNA Mini Kit(方法B)提取HCV RNA,采用实时荧光定量RT-PCR法检测血清HCV RNA载量。取高载量和低载量两份标本,分批重复检测,计算两种方法批内变异系数(CV)。取HCV RNA强阳性质控标本,梯度稀释后检测,评估两种方法检测的敏感度。结果在103例血清抗-HCV阳性血清标本中,经方法A和方法B提取核酸,检测血清HCV RNA阳性率分别为86.4%和82.5%,差异无统计学意义(P>0.05);方法A提取核酸检测的血清HCV RNA载量为(5.4±1.2)lg IU/mL,方法B检测结果为(5.0±1.6)lg IU/mL,差异有统计学意义(t=2.078,P=0.039);方法A提取核酸检测血清HCV RNA载量低值和高值的CV分别为2.4%和2.2%,方法B检测为4.7%和4.5%,方法A提取核酸检测HCV RNA的重复性优于方法B;方法A提取检测血清HCV RNA最低载量为2.8×10^(-6)lg IU/mL,低于方法B检测的3.5×10^(-6)lg IU/mL,表明更灵敏。结论与QIAamp Viral RNA Mini Kit相比,NucliSens miniMag系统检测血清HCV RNA的效能更高。

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.

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.

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.

Rapid and Reliable Method of High-Quality RNA Extraction from Diverse Plants

The isolation of high quality RNA is a crucial technique in plant molecular biology. The quality of RNA determines the reliability of downstream process like real time PCR. In this paper, we reported a high quality RNA extraction protocol for a variety of plant species. Our protocol is time effective than traditional RNA extraction methods. The method takes only an hour to complete the procedure. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The extracted RNA was further used for cDNA synthesis, expression analysis and copy number determination through Real Time PCR. The results indicate that RNA was of good quality and fit for real time PCR. This high throughput plant RNA extraction protocol can be used to isolate high quality RNA from diverse plants for real time PCR and other downstream applications.

Simple Modifications to Standard TRIzol®Protocol Allow High-Yield RNA Extraction from Cells on Resorbable Materials

Resorbable bioceramics are attractive for medical applications such as bone substitution. Biochemical analysis on cells cultured on these biomaterials is vital to predict the impact of the materials in vivo and RNA extraction is an essential step in gene expression study using RT-qPCR. In this study, we describe simple modifications to the TRIzol? RNA extraction protocol widely used in biology and these allow high-yield extraction of RNA from cells on resorbable calcium phosphates. Without the modifications, RNA is trapped in the co-precipitated calcium compounds, rendering TRIzol? extraction method infeasible. Among the modifications, the use of extra TRIzol? to dilute the lysate before the RNA precipitation step is critical for extraction of RNA from porous ?-tricalcium phosphate (?-TCP) discs. We also investigate the rationale behind the undesirable precipitation so as to provide clues about the modifications required for other resorbable materials with high application potential in bone tissue engineering.

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.

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 ＇

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 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.

反转录酶在微小RNA实时荧光定量聚合酶链反应中的作用

目的以微小RNA(miRNA)作为检测样本,考察影响实时荧光定量聚合酶链反应(RT-qPCR)检测结果的关键点,并筛选出最优的检测方法。方法采用3种miRNA提取方法(分别使用EasyPure^(■)miRNA提取试剂盒、miRNA提取试剂盒、TransZol Up Plus RNA提取试剂盒)和3种反转录方法(分别使用TransScript^(■)第一链cDNA反转录试剂盒、Evo M-MLV反转录试剂盒、miRNA第一链cDNA合成试剂盒)处理大鼠纹状体脑区,检测miRNA与c DNA的质量和效率,并使用常规RT-qPCR检测miRNA水平,比较不同方法所得结果。结果3种RNA提取方法得到的miRNA质量和效率比较差异均有统计学意义,其中方法2的效果较好,但方法1、2、3的RT-qPCR结果比较差异无统计学意义(miR-132:25.91±9.79、25.26±10.25、27.28±7.39,miR-U6:27.98±11.25、25.98±9.78、29.62±9.65,均P>0.05);3种反转录方法所得实验结果比较差异有统计学意义,方法3所得结果明显低于方法1、方法2(miR-132:16.53±3.17比35.20±1.06、31.42±2.95,miR-U6:16.63±1.73比36.06±2.57、35.59±1.54,均P<0.05),主要影响RT-qPCR的扩增效率和扩增特异性。结论使用能高效富集约18 nt大小RNA的提取方法和在miRNA的3’末端加多聚A尾(Poly A)的反转录酶,可以得到更可靠的RT-qPCR结果。