RNA的制备及纯度的鉴定

rna制备RNA制备是指通过一系列实验步骤和技术手段，从细胞或组织中提取RNA并纯化，以获得足够纯度和量的RNA样本，供后续实验使用。

RNA制备的过程可以分为细胞裂解、RNA提取和RNA纯化三个步骤。

第一步：细胞裂解细胞裂解是RNA制备的第一步，其目的是使细胞孤立的RNA释放出来。

常见的细胞裂解方法有化学法和物理法两种。

化学法主要是利用细胞壁破裂剂，如SDS、蛋白酶K等，来破坏细胞膜结构，使细胞内容物释放到溶液中，其中包括RNA。

物理法主要是利用高频振荡器、高压破碎仪等机械手段来破坏细胞膜，使细胞内容物释放出来。

第二步：RNA提取RNA提取是RNA制备的核心步骤，其目的是将裂解后的细胞中的RNA从其他生物大分子（如DNA、蛋白质和碳水化合物）中分离出来。

常见的RNA提取方法有酚-氯仿法、硅基膜柱法以及磁珠法等。

酚-氯仿法：该方法主要是利用酚和氯仿的相溶性差异，蛋白质、RNA和DNA在酚-氯仿溶液中的溶解度不同，从而实现RNA的提取。

首先将细胞裂解液加入到等体积的酚醇中，轻轻混合，使蛋白质和DNA被酚沉淀下来。

然后将上清液转移到新离心管中，加入等体积的氯仿，轻轻混合，使RNA在上清液中，DNA、RNA在氯仿相中。

最后进行离心分离，收集上清液中的RNA。

硅基膜柱法：该方法主要是利用硅基膜上的离子交换和疏水相互作用原理，使RNA固定在硅基膜上，而其他杂质则被洗脱下来。

首先将细胞裂解液与硅基膜结合，然后进行洗涤和洗脱步骤，最后收集硅基膜上的RNA。

磁珠法：该方法主要是利用带有磁性的磁珠，结合RNA提取试剂盒中的特定缓冲液，在磁场下将RNA与其他杂质分离。

首先将细胞裂解液与磁珠结合，然后洗涤去除其他杂质，最后在磁场下分离磁珠，将RNA溶于溶剂中。

第三步：RNA纯化RNA纯化是为了去除RNA样品中的杂质，提高RNA的纯度。

主要包括DNaseI消化、乙酰胆碱酯酶消化、酚醇沉淀等方法。

DNaseI消化：该步骤主要是通过在反应体系中加入具有DNaseI活性的酶来去除RNA样品中可能存在的DNA污染。

总RNA 的提取、电泳检测及浓度和纯度的测定撰写人：黄晶一、实验原理1、总RNA的提取见分子克隆（第三版）上册P522 “用一步法从细胞和组织中同时制备DNA、RNA和蛋白质”2、RNA的电泳见分子克隆（第三版）上册P540 “按大小分离RNA: 在含有甲醛的琼脂糖凝胶上进行的RNA电泳”3、RNA的浓度及纯度测定见分子克隆（第三版）下册P1695 “DNA或RNA的分光光度法测定”二、实验准备1、配制0.1%的DEPC水，37℃培养箱内放置过夜，之后灭菌两次。

2、准备大、中、小进口枪头各一盒，1.5ml进口离心管、0.2ml 进口PCR管各一瓶，灭菌两次（公用）3、准备研钵（n）、勺子（n+1）、50ml、100ml量筒各一个，180℃烤4小时以上（n=提取RNA的个数）4、10×MOPS缓冲液、甲醛、30％双氧水、胶布、氯仿（sigma）、异丙醇（sigma）、75％乙醇（以上试剂及物品公用，实验前请确定其是否齐备）三、实验步骤（一）总RNA的提取1．液氮研磨组织，取100mg组织，加入1ml Trizol，室温放置5min2．加入200ul氯仿，剧烈振荡混匀，室温放置3min，之后4℃，12000g,离心15min 3．上清移入新管，加入1/5体积的氯仿，剧烈振荡混匀，室温放置3min, 之后4℃，12000g,离心15min4．上清移入新管，加入等体积的异丙醇，混匀，室温放置10min,之后4℃，12000g,离心15min5．弃上清，加入1ml 75%乙醇，振荡，4℃，7500g,离心5min6．弃上清，室温晾干，之后加入40ul DEPC水溶解（RNA不要完全干燥，否则不利于溶解）（二）RNA的电泳1．用3％-30％的双氧水浸泡RNA专用的电泳槽及胶板、梳子10分钟以上2．之后用0.1%的DEPC水洗涤3．配制1.2%的含有甲醛的琼脂糖凝胶1）称取0.36g RNA专用的琼脂糖，加入24ml DEPC水，熔化后冷却到60℃2）加入3ml 10×MOPS，混匀后再加入3ml甲醛，之后再加入3ul EB4．用DEPC水配制1×MOPS 100ml作为电泳缓冲液，55V 预电泳30-40min5. RNA电泳样品的制备1）将6ul RNA样品、1ul 10×MOPS、1ul甲醛、2ul RNA上样缓冲液混匀2）65℃加热10min,之后冰浴2min,短暂离心后可上样6．60V 电泳30-40min7．紫外检测（三）RNA浓度及纯度的测定1. 运行分光光度计中测定RNA的程序2. 200ul DEPC水作为空白, 5ul RNA样品+295ul DEPC水作为测量样品3. 测量的RNA样品的OD260/280应在1.8-2.0之间4. RNA样品的实际浓度等于分光光度计显示浓度ｘ60四、注意事项1. 整个实验过程中要戴口罩、一次性手套，一次性手套应经常更换，防止污染RNase2. 实验试剂及物品大多数为公用，请养成良好的实验习惯，用毕放回原处，试剂或物品用完或快用完，告知负责人，及时补充3．烤箱不能过夜工作，以免发生火灾4．DEPC为疑似致癌物，操作时应戴口罩、手套，小心吸取，避免污染实验台及物品注：10×MOPS的配制方法0.627g MOPS溶于12ml DEPC水中，加入1.2ml 1M NaAc (DEPC水配制)，用2M NaOH 调pH 7.0, 加入0.75ml 0.2M EDTA, 之后加入DEPC水定容为15ml。

总RNA 的提取、电泳检测及浓度和纯度的测定撰写人：黄晶一、实验原理1、总RNA的提取见分子克隆（第三版）上册P522 “用一步法从细胞和组织中同时制备DNA、RNA和蛋白质”2、RNA的电泳见分子克隆（第三版）上册P540 “按大小分离RNA: 在含有甲醛的琼脂糖凝胶上进行的RNA电泳”3、RNA的浓度及纯度测定见分子克隆（第三版）下册P1695 “DNA或RNA的分光光度法测定”二、实验准备1、配制0.1%的DEPC水，37℃培养箱内放置过夜，之后灭菌两次。

2、准备大、中、小进口枪头各一盒，1.5ml进口离心管、0.2ml 进口PCR管各一瓶，灭菌两次（公用）3、准备研钵（n）、勺子（n+1）、50ml、100ml量筒各一个，180℃烤4小时以上（n=提取RNA的个数）4、10×MOPS缓冲液、甲醛、30％双氧水、胶布、氯仿（sigma）、异丙醇（sigma）、75％乙醇（以上试剂及物品公用，实验前请确定其是否齐备）三、实验步骤（一）总RNA的提取1．液氮研磨组织，取100mg组织，加入1ml Trizol，室温放置5min2．加入200ul氯仿，剧烈振荡混匀，室温放置3min，之后4℃，12000g,离心15min 3．上清移入新管，加入1/5体积的氯仿，剧烈振荡混匀，室温放置3min, 之后4℃，12000g,离心15min4．上清移入新管，加入等体积的异丙醇，混匀，室温放置10min,之后4℃，12000g,离心15min5．弃上清，加入1ml 75%乙醇，振荡，4℃，7500g,离心5min6．弃上清，室温晾干，之后加入40ul DEPC水溶解（RNA不要完全干燥，否则不利于溶解）（二）RNA的电泳1．用3％-30％的双氧水浸泡RNA专用的电泳槽及胶板、梳子10分钟以上2．之后用0.1%的DEPC水洗涤3．配制1.2%的含有甲醛的琼脂糖凝胶1）称取0.36g RNA专用的琼脂糖，加入24ml DEPC水，熔化后冷却到60℃2）加入3ml 10×MOPS，混匀后再加入3ml甲醛，之后再加入3ul EB4．用DEPC水配制1×MOPS 100ml作为电泳缓冲液，55V 预电泳30-40min5. RNA电泳样品的制备1）将6ul RNA样品、1ul 10×MOPS、1ul甲醛、2ul RNA上样缓冲液混匀2）65℃加热10min,之后冰浴2min,短暂离心后可上样6．60V 电泳30-40min7．紫外检测（三）RNA浓度及纯度的测定1. 运行分光光度计中测定RNA的程序2. 200ul DEPC水作为空白, 5ul RNA样品+295ul DEPC水作为测量样品3. 测量的RNA样品的OD260/280应在1.8-2.0之间4. RNA样品的实际浓度等于分光光度计显示浓度ｘ60四、注意事项1. 整个实验过程中要戴口罩、一次性手套，一次性手套应经常更换，防止污染RNase2. 实验试剂及物品大多数为公用，请养成良好的实验习惯，用毕放回原处，试剂或物品用完或快用完，告知负责人，及时补充3．烤箱不能过夜工作，以免发生火灾4．DEPC为疑似致癌物，操作时应戴口罩、手套，小心吸取，避免污染实验台及物品注：10×MOPS的配制方法0.627g MOPS溶于12ml DEPC水中，加入1.2ml 1M NaAc (DEPC水配制)，用2M NaOH 调pH 7.0, 加入0.75ml 0.2M EDTA, 之后加入DEPC水定容为15ml。

rna实验报告RNA 实验报告一、实验目的本次 RNA 实验旨在提取和分析细胞中的 RNA，以了解 RNA 的特性、结构和功能，并掌握相关实验技术和方法。

二、实验原理RNA 是一种重要的生物大分子，在基因表达和调控中起着关键作用。

RNA 提取的基本原理是利用细胞裂解液破坏细胞膜和核膜，使RNA 释放出来，然后通过酚/氯仿抽提、乙醇沉淀等步骤将 RNA 从其他细胞成分中分离出来。

RNA 的质量和纯度可以通过紫外分光光度计测定吸光度比值（A260/A280 和 A260/A230）来评估，完整性可以通过琼脂糖凝胶电泳进行检测。

三、实验材料与设备1、材料细胞样本（如培养的细胞系或组织样本）液氮无水乙醇氯仿异丙醇RNA 提取试剂盒DEPC 处理水2、设备冷冻离心机移液器恒温水浴锅紫外分光光度计琼脂糖凝胶电泳系统四、实验步骤1、细胞裂解将细胞样本收集到离心管中，加入适量的裂解液，充分混匀，使细胞完全裂解。

2、抽提加入等体积的酚/氯仿，剧烈振荡混匀，室温静置 5 分钟，然后在 4℃下以 12000 rpm 离心 15 分钟。

3、沉淀 RNA将上清液转移到新的离心管中，加入等体积的异丙醇，轻轻混匀，室温静置 10 分钟，然后在 4℃下以 12000 rpm 离心 10 分钟。

4、洗涤 RNA弃上清液，加入 75%的乙醇，轻轻混悬沉淀，在 4℃下以 7500 rpm 离心 5 分钟。

5、溶解 RNA弃上清液，晾干沉淀，然后用适量的 DEPC 处理水溶解 RNA。

6、 RNA 质量和纯度检测使用紫外分光光度计测定 RNA 溶液在 260nm、280nm 和 230nm 处的吸光度，计算A260/A280 和A260/A230 的比值，评估RNA 的纯度。

7、 RNA 完整性检测配制 1%的琼脂糖凝胶，将 RNA 样品与上样缓冲液混合后进行电泳，观察 28S、18S 和 5S RNA 条带的清晰度和完整性。

五、实验结果1、 RNA 质量和纯度检测结果测定的 A260/A280 比值在 18 20 之间，A260/A230 比值大于 20，表明提取的 RNA 纯度较高，无蛋白质和有机溶剂等杂质污染。

RNA的质量和纯度鉴定2011年02月22日星期二04:17摘自RNA:A Laboratory Manual,by Donald C.Rio,Manuel Ares Jr,Gregory J.Hannon,and Timothy W.Nilsen.CSHL Press,Cold Spring Harbor,NY,USA,2010.RELATED INFORMATIONSeveral methods for RNA purification are described in Purification of RNA by SDS Solubilization and Phenol Extraction(Rio et al.2010a)and Ethanol Precipitation of RNA and the Use of Carriers (Rio et al.2010b).RNA samples contaminated with DNA can be purified using DNAse I,as described in Removal of DNA from RNA(Rio et al.2010c).RNA molecules≤600kb can be analyzed using polyacrylamide gel electrophoresis(Polyacrylamide Gel Electrophoresis of RNA [Rio et al.2010d]),whereas larger RNA molecules such as messenger RNA(mRNA)are more effectively analyzed on agarose gels(Nondenaturing Agarose Gel Electrophoresis of RNA[Rio et al.2010e]).A protocol is also available for Northern Hybridization(Sambrook and Russell2006). Before attempting to use any of the following procedures for DNA quantitation,it is important to have a"ballpark"idea of what you expect the yield to be(see Table1).DETERMINING YIELD BY SPECTROPHOTOMETRY OR FLUORIMETRYThe easiest way to determine the quantity of RNA in a sample is to measure the absorbance at 260nm(A260)using a spectrophotometer.Because the bases in RNA absorb ultraviolet(UV)light in the250-to265-nm range,one can use this property to quantitatively measure the concentration of an RNA solution,using an average absorbance for the four nucleotide bases.A solution of RNA at40µg/mL will have an absorbance of~1.Accordingly,if50µL of the same solution is diluted in1mL of H2O and read in a1-mL cuvette,the absorbance will be0.05;this is the absolute minimum that we recommend for accurate optical density(OD)readings.The advent of nanospectrophotometers,such as the Thermo-Fisher NanoDrop and GE Healthcare NanoVue,has greatly increased the ease,sensitivity,and accuracy of determining the concentration of low-volume(microliters)samples by UV absorbance(see below).With this equipment,one can measure the A260without dilution and with minimal waste of the sample.If you do not have access to these spectrophotometers,we recommend that OD be used as a measure of concentration only when you have a significant amount of RNA present in a concentrated solution,e.g.,at least1.0µg/µL.We have often observed students interpret A260 readings of0.003or less,but measurements as low as this are essentially meaningless. Although measuring A260is generally a reliable way to quantitate RNA concentrations,this method can be confounded if the RNA is contaminated with DNA,protein,or phenol,all of which absorb some UV light at260nm.A diagnostic for protein contamination is absorbance at280nm. Phenol and TRIzol Reagent both absorb at270nm and230nm.The chaotropic agents guanidine-HCl and guanidinium isothiocyanate,commonly used for RNA purification,absorb at ~230nm and~260nm,respectively.For purified DNA the A260:A280ratio should be~1.8,and for purified RNA this ratio should be~2.0,because the unpaired bases in RNA absorb more UV light than the base-paired bases in duplex DNA.However,these ratios are"rules of thumb"and assume an average base composition of the RNA sample,because different bases each have different A260:A280ratios.If the A260:A280ratio is<2.0,protein contamination is probable and re-extraction with phenol is recommended.If the A260is high,phenol contamination is probableand another round of ethanol precipitation and resuspension is recommended. Contamination with DNA is harder to detect because RNA and DNA essentially have identical absorbance spectra.Therefore,if the A260of the sample is higher than expected and protein and phenol contamination have been ruled out,the sample is likely contaminated with DNA.In this case,DNase treatment followed by phenol extraction and ethanol precipitation is recommended (see Purification of RNA by SDS Solubilization and Phenol Extraction[Rio et al.2010a]and Ethanol Precipitation of RNA and the Use of Carriers[Rio et al.2010b]).In addition,it is possible to use the A260:A230ratio as an indicator of nucleic acid purity,with ratios commonly in the range of2.0-2.2.Note that absorbance is pH dependent,so for accurate readings,keep the pH constant and near7.5.UV Absorbance Determination of RNA Concentrations Using a NanospectrophotometerSmall-volume(0.5-2µL)UV-visible spectrophotometers can now be used to sensitively measure RNA or DNA concentrations and fluorochrome(e.g.,Cy3/Cy5)dye coupling to allylamine-modified cDNA,or for protein concentration determination.These instruments(e.g., the Thermo-Fisher NanoDrop or GE Healthcare NanoVue nanospectrophotometers)use fiber optic technology and surface tension to hold a1-µL sample in place;thus,they eliminate the need for traditional sample holding by cuvettes.The dynamic range of these instruments is high, ranging from2ng/µL to3700ng/µL.The following discussion provides practical information about the use of this equipment and interpretation of results.Step-by-step instructions for operation are supplied in the manufacturers’instrument manuals.ProcedureThis procedure is simple.Place1.0-1.5µL of the RNA sample onto the sample pedestal.The UV absorbance of the sample is then read either at a fixed wavelength or in a UV-visible scan. Typically,pure RNA(or DNA)samples are read at A260and A280.It is possible also to use these instruments to scan a full UV and visible wavelength absorbance spectrum,from220nm to750 nm.The UV-visible wavelength scanning procedure is more useful in microarray studies when one wants to quantitate fluorochrome dye coupling to cDNA.Figure1shows some results obtained using a nanospectrophotometer and how these results are interpreted.Tips and Troubleshooting1.As with the UV absorbance methods described previously,any compounds(e.g.,free nucleotides,phenol,or other organic compounds such as guanidinium isothiocyanate)that absorb UV light near260nm will interfere.Often,a simple ethanol precipitation can be used to remove the contaminating substance.2.When adding a sample to the pedestal,ensure that the sample is placed over the"eye"(the eye is the little metal circle with the tiny hole in the NanoDrop).3.Bubbles are incompatible with accurate readings.Although the instructions may claim that only1µL is needed,in practice a larger volume(1.3-1.5µL)will produce more reliable readings because the droplet will have better optical characteristics.Small-volume droplets can give incomplete coverage across the pedestal.4.It is possible to saturate the spectrophotometer with high-concentration solutions of RNA or DNA.This will cause an underestimate of the true concentration.Try to obtain readings using solutions at≤2.5µg/µL.For samples that give higher readings(>2.5µg/µL),it is a good idea to dilute1:10and read the dilution to get the most accurate reading.Fluorescent Dye Binding for RNA and DNA QuantitationAs an alternative to spectrophotometry,RNA can be quantitated using fluorescent dye binding. This is a sensitive assay for detecting and determining the quantity of RNA(and contaminating DNA)present in a purified RNA sample or in crude extracts or chromatographic fractions.It is ~1000times more sensitive than using UV absorbance and can detect RNA at1ng/mL.In addition,this method can be used for quantitation of in vitro-transcribed RNA samples or for determining RNA concentrations before Northern blotting,RNase protection,reverse transcriptase polymerase chain reaction(RT-PCR),or cDNA library preparation.It is similar in concept to fluorescent dye binding to DNA(e.g.,SYBR Green)used in quantitative PCR(qPCR). The RiboGreen RNA reagent,a proprietary fluorescent dye,preferentially binds to RNA,but it can also detect DNA(see the following"Tips and Troubleshooting"section).This method is useful when making RNA from nuclear fractions that might be contaminated with DNA and for assaying very small quantities of RNA prepared from limited quantities of starting material.Step-by-step instructions for using this reagent are supplied in the manufacturer’s user manual.Tips and Troubleshooting1.If you suspect that the RNA sample of interest is contaminated with DNA(perhaps cellular genomic DNA in a total RNA preparation),you can treat the sample with DNase I(Removal of DNA from RNA[Rio et al.2010c])or use PicoGreen,which detects double-stranded DNA only.2.The assay remains linear in the presence of several compounds that commonly contaminate nucleic acid preparations,although the signal intensity may be affected.Thus,to serve as an effective control,treat the RNA solution used to prepare the standard curve the same way as the experimental samples;it should contain similar levels of such compounds.3.There can be some interference with the fluorescence assay by salts,organic solvents, detergents,proteins,or other compounds.If these are present in the sample,control for this by placing them in the standard curve RNA dilutions.DETERMINING YIELD BY GEL ELECTROPHORESISFor samples of total or cytoplasmic RNA(with ribosomal RNA[rRNA]),a simple and straightforward way to determine yield is to separate a small aliquot of the RNA on an agarose gel and stain with ethidium bromide or SYBR Gold(see Nondenaturing Agarose Gel Electrophoresis of RNA[Rio et al.2010e]).Bands of rRNA(28S and18S)are visualized,and their intensity is compared with that of a preparation of known quantity.In our laboratories,we keep a "stock"of high-quality cytoplasmic RNA prepared from tissue culture cells as a reference.This stock can be diluted as appropriate to obtain the equivalent amount of RNA that you expect from the sample.Because SYBR Gold is10-fold more sensitive than ethidium bromide,it should be used for small amounts of RNA.An advantage of this technique is that it measures both the quantity and the quality of RNA;i.e., sharp and distinct rRNA bands without a pronounced haze below them is a good sign that the preparation is not significantly degraded.The only disadvantage of this approach is sensitivity;at least500ng of RNA must be available to sacrifice for this assessment.To assess quality,sufficient quantity must be loaded to detect degraded material.Use of Rapid Capillary Electrophoresis on an Agilent Bioanalyzer for RNA Sample Quality Control A bioanalyzer provides a convenient and more sensitive way to assess RNA quality.The Agilent2100Bioanalyzer basically performs small-volume electrophoresis,similar to capillary DNA sequencing but in a"chip"format that allows analysis of12samples at once.The bioanalyzer can be used for analysis of the quality of total RNA preparations by visualizing the rRNA bands and intact mRNA,and to detect and quantify RT-PCR products(using DNA chips;see Klinck et al. 2008;Venables et al.2008,2009).Agilent RNA kits,which are designed for use with the Agilent 2100Bioanalyzer only,contain chips and reagents designed for analysis of RNAs.Each RNA chip contains an interconnected set of microchannels(capillaries)that is used for separation of nucleic acid fragments based on their size as they are driven through the microchannels electrophoretically.The following discussion provides practical information about the use of this equipment and interpretation of results obtained running RNA samples on a bioanalyzer. Step-by-step instructions for operation of this equipment are supplied in the manufacturer’s user manuals.A computer runs the machine,analyzes the traces,and presents the data.Of interest is the trace that estimates the amount of each rRNA that is present and their ratios.Because the larger rRNA is more susceptible to degradation due to its greater length,the ratio of28S to18S rRNA is a convenient measure of the integrity of the sample.This information is displayed by the software. The amount of RNA in the sample is determined by comparison to a fixed known amount of the RNA ladder RNAs.Thus,it is possible to obtain both concentration and integrity of the sample. Because the data are simple to archive and compare,many laboratories that handle and compare many samples believe that this approach is superior to gel analysis,primarily because of its data archiving and comparison ability.Expected Results of Bioanalyzer Analysis of RNA QualityThe quality of purified total RNA can be analyzed using a bioanalyzer,such as the Agilent2100.In Figure2,this bioanalyzer was used to show the presence of18S and28S rRNA(mammalian),18S and25S/26S rRNA(yeast),16S and23S rRNA(bacteria),and small RNA species in total RNA. Typically,for total RNA samples,two major rRNA peaks are observed,because these account for >90%of the total RNA in cells.For eukaryotic total RNA,the two rRNA peaks correspond to the 18S and28S rRNAs;for yeast total RNA,the two large rRNA peaks correspond to the18S and 25S/26S rRNAs;for bacterial total RNA,the two large rRNA peaks correspond to the16S and23S rRNAs(see Fig.2).Tips and Troubleshooting1.Sample preparation for the RNA Nano Chip:i.Ideally,sample concentrations should be100-200ng/µL;RNA concentrations as low as50 ng/µL can be used.ii.Each sample well must contain a total of6µL(1µL for the RNA Pico Chip).iii.The nano marker must be placed in every sample well and the ladder well.iv.Add water or nano marker to unused wells to bring the volume up to6µL.e the chip within5min of preparation to prevent evaporation.Cover the chip with plastic wrap or Parafilm if it will be left standing for any length of time.vi.RNA samples may be denatured to remove secondary structure.Denature for2min at 70°C before placing the samples in the wells of the chip.vii.Genomic DNA can produce stray bands or clog the capillaries in the chip.To check for genomic DNA contamination,treat the samples with DNase I.Run a DNase-I-treated sample next to an untreated sample.2.It is important not to leave the used chip in the bioanalyzer after the run is complete,because this will dry out the electrodes and make them difficult to clean.3.A critical part of the assay is preparing the chip.This involves loading the capillaries with the "gel"matrix material and the dyes that will stain the RNA so that the detector in the machine can measure it.The chip is loaded with a syringe system that is fairly easy to use.Fill each well(make sure that there are no bubbles!),add the sample,vortex,and load into the machine according to the instructions.4.Because this protocol uses small-volume electrophoresis,the samples must be in a low-ionic-strength solution(preferably in RNase-free water or10mM TE buffer).5.Accurate pipetting is very important for reproducible results.Make sure to use properly calibrated pipettes and to place the tip into the center and bottom of each well in the chip when dispensing.To avoid bubbles,do not push past the first resistance point on the pipette.You may pipette up and down gently to mix samples in the wells of the chip.6.Protect the gel-dye mix from light by covering the tube with foil.Return the reagents to the cold room when you are finished.7.When using the priming station,press down slowly and steadily on the plunger when priming.After releasing,the plunger should come up to at least0.7mL in1-2sec.If this does not occur,check that the gasket is clean and retry.If it still does not prime well,change the gasket (see Agilent2100instrument manual).DETERMINING YIELD BY QUANTITATIVE OR SEMIQUANTITATIVE PCRWhen it is not possible to quantitate the amount of RNA in a preparation by any of the methods previously described(e.g.,when the amount of expected RNA is very small),reverse transcription followed by qPCR or semi-qPCR is recommended.These techniques are extremely sensitive and provide information with regard to both the quantity and quality of RNA.In addition,these approaches allow a determination of whether the sample is significantly contaminated with DNA. We recommend assaying a housekeeping mRNA(e.g.,actin,glyceraldehyde3-phosphate dehydrogenase[GAPDH],tubulin,etc.).Again,as with gel electrophoresis,we recommend using a"stock"RNA preparation(diluted as appropriate)as a reference.Parallel assessment with and without reverse transcription will show any DNA contamination(i.e.,amplification without reverse transcription indicates the presence of DNA,unless the PCR primer pairs are in different exons separated by a large intron;in this case,the genomic DNA contamination product will be too large to show up).For RNA preparations in which large RNAs have been removed,it is necessary to assay a small RNA(e.g.,a constitutively expressed micro RNA[miRNA])by RT-PCR. All of the techniques described above are useful methods for assessing the quantity of RNA that has been prepared;gel electrophoresis and qPCR or semi-qPCR also provide information regarding mon sense indicates that if the yield is far below(≥30%)that expected, something has gone wrong with the preparation and the RNA is suspect.Rather than proceeding with a suspect preparation,start over from the beginning.NORTHERN BLOTTING TO ASSESS THE QUALITY OF RNA PREPARATIONSAs discussed previously,gel electrophoresis,visualization of RNA bands,and qPCR or semi-qPCR (in all cases compared with a reference)are valuable methods for assessing quantity as well as quality of RNA.Nevertheless,each of these methods can be potentially misleading.PCR will amplify fragments of RNA,and rRNA visualization is a somewhat crude method for assessingintegrity of an RNA preparation.Moreover,spectrophotometry tells nothing about the integrity of the RNA preparation.By far,the best method for assessing the quality of an RNA preparation is Northern blotting(for an example,see Northern Hybridization[Sambrook and Russell2006]). Because this technique visualizes the entire RNA,it is diagnostic for any degradation.Therefore,if you are working with specific RNAs,we recommend assaying each preparation by Northern blotting.Again,this is best done by comparing the quality(i.e.,sharp band)and quantity of the signal obtained to those obtained from a reference Northern blot performed on RNA of known quantity and quality.WEB RESOURCESNanospectrophotometry/(GE Healthcare NanoVue User Manual28944299AA)/Support.aspx?Type=User%20Guides&Cat=NanoDrop%201000 (NanoDrop User Bulletin T042,Nucleic Acid Purity Ratios)/(Thermo Fisher Scientific,Inc.NanoDrop User Manual[nd-1000-v3.7 User’s Manual])Fluorescent Dye Binding/site/us/en/home/References/Molecular-Probes-The-Handbook/Nucl eic-Acid-Detection-and-Genomics-Technology/Nucleic-Acid-Detection-and-Quantitation-in-Soluti on.html/(Quant-iT RiboGreenRNA Reagent and Kit:mp11490)Bioanalyzer/(Agilent2100Bioanalyzer User Guide;Agilent RNA6000Nano Kit Guide G2938-90034_KitRNA6000Nano_ebook;Quantitation comparison of total RNA using the Agilent2100bioanalyzer,RiboGreen analysis and UV spectrometry;Agilent2100Bioanalyzer User Guide)REFERENCES1.Applied Biosystems.2010.RNA yields from tissues and cells.Appplied Biosystems,Austin, TX,/techlib/append/rna_yields.html.[Abstract/Free Full Text]2.Klinck R,Bramard A,Inke L,Dufresne-Martin G,Gervais-Bird J,Madden R,Paquet ER,Koh C, Venables JP,Prinos P,et al.2008.Multiple alternative splicing markers for ovarian cancer.Cancer Res68:657–663.[Abstract/Free Full Text]3.Lightfoot S.2002.Quantitation comparison of total RNA using the Agilent2100bioanalyzer, ribogreen analysis and UV spectrometry.Agilent Technologies,Palo Alto,CA, /en-US/Search/Library/_layouts/Agilent/PublicationSummary.aspx ?whid=30152&liid=780.[Abstract/Free Full Text]4.Qiagen.2006.RNeasy mini handbook.Qiagen,Germantown,MD, /literature/render.aspx?id=352.[Abstract/Free Full Text]5.Rio DC,Ares M Jr,Hannon GJ,Nilsen TW.2010a.Purification of RNA by SDS solubilization and phenol extraction.Cold Spring Harb Protoc(this issue).doi: 10.1101/pdb.prot5438.[Abstract/Free Full Text]6.Rio DC,Ares M Jr,Hannon GJ,Nilsen TW.2010b.Ethanol precipitation of RNA and the use of carriers.Cold Spring Harb Protoc(this issue).doi:10.1101/pdb.prot5440.[Abstract/Free Full Text]7.Rio DC,Ares M Jr,Hannon GJ,Nilsen TW.2010c.Removal of DNA from RNA.Cold Spring Harb Protoc(this issue).doi:10.1101/pdb.prot5443.[Abstract/Free Full Text]8.Rio DC,Ares M Jr,Hannon GJ,Nilsen TW.2010d.Polyacrylamide gel electrophoresis of RNA. Cold Spring Harb Protoc(this issue).doi:10.1101/pdb.prot5444.[Abstract/Free Full Text]9.Rio DC,Ares M Jr,Hannon GJ,Nilsen TW.2010e.Nondenaturing agarose gel electrophoresis of RNA.Cold Spring Harb Protoc(this issue).doi:10.1101/pdb.prot5445.[Abstract/Free Full Text]10.Sambrook J,Russell DW.2006.Northern hybridization.Cold Spring Harb Protoc doi:10.1101/pdb.prot3723.[Free Full Text]11.Venables JP,Koh CS,Froehlich U,Lapointe E,Couture S,Inkel L,Bramard A,Paquet ER, Watier V,Durand M,et al.2008.Multiple and specific mRNA processing targets for the major human hnRNP proteins.Mol Cell Biol28:6033–6043.[Abstract/Free Full Text]12.Venables JP,Klinck R,Koh C,Gervais-Bird J,Bramard A,Inkel L,Durand M,Couture S, Froehlich U,Lapointe E,et al.2009.Cancer-associated regulation of alternative splicing.Nat Struct Mol Biol16:717–724.[Medline]。

rna的工艺制备流程RNA是核糖核酸的缩写，属于生物分子中的一种。

它起到了DNA的传递和执行遗传信息的功能，在基因表达中扮演着重要角色。

那么，如何制备RNA呢？下面将为大家介绍RNA的工艺制备流程。

RNA的工艺制备流程可以分为以下几个步骤：第一步，收集需要制备RNA的样本。

这个样本可以是从动植物组织、细菌、真菌、病毒等生物中提取得到。

样本的选择需要根据实验的目的和具体需求来确定。

第二步，进行细胞裂解。

细胞裂解是将收集到的样本中的细胞破碎，释放出内部的RNA。

常用的方法有机械裂解、超声波裂解和化学裂解等。

裂解完毕后，可以利用离心技术分离细胞碎片和细胞液。

第三步，去除DNA和蛋白质。

在这个步骤中，可以通过加入DNA 酶和蛋白酶等酶类来分解DNA和蛋白质。

这样可以保证得到的RNA纯度高。

第四步，利用柱层析等纯化技术纯化RNA。

纯化过程中可以根据RNA的特性使用离子交换柱、凝胶过滤柱等进行分离。

通过纯化步骤可以得到高纯度的RNA样品。

第五步，进行RNA的逆转录反应。

逆转录反应是将RNA转录为相应的DNA，这一步可以利用逆转录酶进行。

逆转录反应后可以得到cDNA。

第六步，PCR扩增。

利用cDNA作为模板进行PCR反应，可以扩增出想要的特定片段。

这一步可以根据实际需求进行调整，可以进行常规PCR、荧光定量PCR等。

第七步，测定RNA的浓度和纯度。

通过光谱仪测定RNA的吸光度，可以得到RNA的浓度，并通过260nm/280nm比值判断其纯度。

最后一步，保存RNA样本。

将制备得到的RNA样本进行冷冻保存，可以使其长期保持在-80°C下的稳定状态，方便后续的分子生物学实验。

总的来说，RNA的工艺制备流程包括样本收集、细胞裂解、去除DNA和蛋白质、纯化RNA、逆转录反应、PCR扩增、测定浓度和纯度以及保存。

这一流程在实验室中得到广泛应用，能够帮助科学家们深入研究RNA的结构和功能，为基因表达和遗传研究提供有力支持。