熒光定量PCR實(shí)驗(yàn)設(shè)計(jì)及優(yōu)化_lxm概要

1、熒光定量PCR實(shí)驗(yàn)設(shè)計(jì)及優(yōu)化picture placeholder2PCR的建立與優(yōu)化核酸純化與逆轉(zhuǎn)錄PCR試劑體系引物、探針的設(shè)計(jì)33長(zhǎng)度 16 - 24 bases（引物） 序列 不能有以下情況：任意2個(gè)引物或探針之間的序列互補(bǔ)引物或探針本身有二級(jí)結(jié)構(gòu)連續(xù)的 GGG，引物的5端有G引物3端最后5個(gè)堿基有2個(gè)以上的G或C引物3端最后1個(gè)堿基為A GC含量 40 - 60 % GC GC/AT分布大致均衡,C含量G探針復(fù)性溫度 6570C 引物復(fù)性溫度 6065C PCR引物與探針4標(biāo)記 淬滅基團(tuán)采用無(wú)熒光背景的如BHQ, 不要用tamraPCR產(chǎn)物長(zhǎng)度 50150bpDesign prime

2、r design software，選擇序列保守區(qū)域 如果保守區(qū)域太短，可以在引物中考慮引入LNA提高Tm 探針可以考慮引入LNA或改用MGB或自淬滅taqman探針 簡(jiǎn)并引物或探針需要同時(shí)考慮多種情況下的Tm高低純化與否 PAGE/ HPLCPCR引物與探針5SNP的Taqman雙色檢測(cè)中，突變探針一般Fam標(biāo)記相對(duì)定量分析中內(nèi)參基因一般Hex標(biāo)記 (例如Roche的UPL相對(duì)定量解決方案) 對(duì)檢測(cè)要求更高的建議Fam標(biāo)記(靈敏度或者 定量 vs 定性) 擴(kuò)增效率較差的建議Fam標(biāo)記 PCR引物與探針6Reaction ConditionsPrimers and ProbesConcentr

3、ation Range (final con.)Primers:0.1 M 1.0 M eachProbes:0.1 M 0.5 MStandard Concentrations (final conc.)Primers:0.5 M eachProbes:0.2 MExample: Universal ProbeLibrary assayHigher primers/probe concentrations give better signal to noise ratio (higher fluorescence signal intensitiy)Only minor influence

4、on Cp when used in medium concentration ranges7PCR的建立與優(yōu)化核酸純化與逆轉(zhuǎn)錄PCR試劑體系引物、探針的設(shè)計(jì)8建立新的PCR體系: General Hints模板 - DNA或 cDNA 為模板對(duì)照 - NTC (no template control) 每個(gè)探針引物組合- 陽(yáng)性對(duì)照 (如果可能)分析 - 擴(kuò)增: sensitivity and efficiency of PCR (crossing points)- 熔解曲線分析 (SYBR Green I format):檢測(cè)引物二聚體或其它非特異性擴(kuò)增凝膠電泳: 驗(yàn)證非特異性擴(kuò)增 melt

5、ing curve (雜交探針等模式): 突變檢測(cè)或亞型分析9Standard Conditions in PCRMgCl2 Concentration: 2 - 5 mM10Basic Optimization in PCRUse any template nucleic acid (NA) suitable for PCR, sufficiently purified andfree of PCR inhibitors Template Concentration:建議測(cè)試多個(gè)稀釋度 (最少2個(gè)梯度,102) genomic DNA50 ng - 5 pg plasmid DNAappro

6、x. 106 copies RNA 500 ng total RNA or 100 ng mRNA cDNA 50 ng equivalent of total RNA ，RT product undiluted, 1:10 and 1:100 dilutionsNA StorageStore nucleic acids in high concentrations and aliquotsFor low template concentrations, use siliconized tubes and addcarrier NA (10 ng/l), e.g. MS2 RNA 11Basi

7、c Optimization:MgCl2 Titration MgCl2 Titration: 2 - 5 mM Format: SYBR Green I LC FastStart DNA Master12Basic Optimization:Template Concentration 模板濃度過(guò)高會(huì)抑制PCR Template DNA undiluted and 1:10 dilution Format SYBR Green I LIghtCycler DNA Master NTCsample 1 originalsample 1 1:1 0 sample 2 originalsample

8、 2 1:1013SYBR Green I FormatPCR Analysis Example 模板拷貝數(shù)越低，越容易出現(xiàn)引物二聚體QuantificationMelting Curve Analysis14Optimization:Influence of PCR InhibitorsPCR抑制物可通過(guò)適當(dāng)稀釋樣品降低對(duì)其對(duì)PCR的影響NTCcDNA originalcDNA 1: 2 cDNA 1: 4cDNA 1: 20 Template cDNA undiluted, 1:2, 1:4, 1:20 dilutions Format SYBR Green I LightCycler D

9、NA Master15Further Optimization in PCR檢測(cè)靈敏度和信號(hào)強(qiáng)度可以通過(guò)調(diào)整下列參數(shù)得以改進(jìn)16PCR的建立與優(yōu)化核酸純化與逆轉(zhuǎn)錄PCR試劑體系引物、探針的設(shè)計(jì)AAAAAAAAAAAAtRNAmRNArRNARNase不同豐度組織名稱(chēng)樣品量總RNA含量高豐度肝臟1mg2-4g脾臟1mg心臟1mg中豐度大腦1mg 0.05-2g胚胎1mg腎臟1mg肺1mg低豐度膀胱1mg 0-0.05g骨1mg脂肪1mg高豐度成纖細(xì)胞1050.5-1g人白細(xì)胞105細(xì)胞中RNA的種類(lèi)和含量“chaotropic salts” in binding bufferRoche 手工提取

10、RNA的試劑盒細(xì)胞和組織High Pure RNA Isolation KitHigh Pure RNA Tissue KitTriPure Isolation Reagent甲醛固定石蠟包埋組織和石蠟包埋組織High Pure FFPE RNA Micro KitHigh Pure RNA Paraffin Kit提取mRNAmRNA Isolation KitmRNA Capture KitHigh Pure miRNA Isolation Kit病毒 RNAHigh Pure Viral RNA Kit*High Pure Viral Nucleic Acid KitRNA操作注意事項(xiàng)R

11、NA酶（Rnase）是導(dǎo)致RNA降解最主要的物質(zhì)。此酶非常穩(wěn)定，在一些極端的條件下只可暫時(shí)失活，但限制因素去除后又迅速恢復(fù)活性。常規(guī)高溫高壓滅菌方法和蛋白抑制劑不能使所有的Rnase完全失活。1. Rnase 廣泛存在于人的皮膚上，因此制備RNA時(shí)必須戴手套2. Rnase 可存在于取液器中，因此設(shè)置RNA 專(zhuān)用pipettor, 或者采用以DEPC配制的 70%乙醇擦洗取液器的內(nèi)部和外部，可基本達(dá)到要求。3.塑料制品、玻璃和金屬物品的處理a. 盡量已標(biāo)明RNase-free的塑料制品，如沒(méi)有開(kāi)封使用過(guò)，通常不必處理， 處理步驟：在浸泡塑料制品的容器中，加入終濃度為0.05%0.1% 的DEP

12、C-H2O，室溫放置過(guò)夜，第二天將DEPC-H2O倒出，高溫高壓蒸汽滅菌至少30分鐘。 b. 玻璃和金屬物品250烘烤3小時(shí)以上或者180烘烤8小時(shí)以上RNA操作注意事項(xiàng) 選擇新鮮血液，不得超過(guò)4小時(shí) 選擇新鮮、生長(zhǎng)旺盛的組織 選擇處于生長(zhǎng)旺盛時(shí)期的細(xì)胞可將新鮮血液先進(jìn)行紅細(xì)胞裂解，得到白細(xì)胞，然后加入一定量的細(xì)胞裂解液/TRNzol，-70保存較長(zhǎng)時(shí)間 推薦使用專(zhuān)門(mén)的RNA樣品儲(chǔ)存液進(jìn)行儲(chǔ)存 去掉培養(yǎng)基，加入一定量RNA 提取試劑，-70保存較長(zhǎng)時(shí)間RNA 應(yīng)分裝后凍存于-70冰箱，樣品不能反復(fù)凍融長(zhǎng)期保持：RNA提取步驟進(jìn)行到在70%乙醇洗滌時(shí)，停止實(shí)驗(yàn)，讓RNA保存于70% 乙醇中RNA

13、的評(píng)價(jià)與鑒定純度（ OD260 /OD280 ）：OD260 /OD280 2.1說(shuō)明有部分降解得率（OD260） Yield OD260稀釋倍數(shù)40ng/ul紫外分光光度計(jì)檢測(cè)1瓊脂糖，6V/cm，15min，上樣13 ul 電泳檢測(cè)M 1 2 M 1 2DNA殘留蛋白殘留RNA的評(píng)價(jià)與鑒定23RT反應(yīng)主要組成成分Reverse Transcriptase AMV Reverse Transcriptase (Avian Myeloblastosis Virus) 48 55C M-MuLV Reverse Transcriptase (Moloney Murine Leukaemia) 3

14、7 42C Transcriptor Reverse Transcriptase (recombinant, E. coli. ) up to 65C引物（Oligo dT or Random Primers or Gene Specific Primers) Sequence-specific primers Random Hexamer Primers and anchored oligo(dT)NRNA模板24Transcriptor Reverse TranscriptaseTranscriptor reverse transcriptaseAbility to synthesize

15、fragments up to 14 kb (full length cDNA)Reverse transcription of difficult templates (e.g., GC-rich RNA templates) due to thermostability up to 65CRNase H activity: degrades RNA in RNA:DNA hybridTranscriptor RTInvitrogen Superscript IIInvitrogen Superscript IIIMax Product size14 kb12.3 kb12.3 kbReac

16、tion time30 mins50 mins30-60minReaction temperature42-65 42 50-55 Sensitivity50pg total RNA1ng total RNA10pg RNAGC-rich templateYESNo infoNo infoRNase H activityYESReducedReduced25一步法RT-PCR VS. 二步法 RT-PCR 2-step RT-PCRreverse transcription and PCR-amplification separately1-step RT-PCRreverse transcr

17、iption and PCR- amplification in one reactioneverything for reverse transcriptioneverything for PCR108 copies of the sequence of interesteverything for reverse transcriptionNewly synthesized cDNAcDNA together with everything for PCR108 copies of the sequence of interest26一步法和二步法 RT-PCR優(yōu)點(diǎn)比較一步法RT-PCR的

18、優(yōu)點(diǎn)二步法 RT-PCR的優(yōu)點(diǎn)降低污染的風(fēng)險(xiǎn)Single tube ReactionNo transfer /opening requiredAutomation is possible可以分別優(yōu)化RT和PCR反應(yīng)條件Efficient and accurate amplification增加了靈敏度和特異性Sequence-specific primersEntire cDNA sample as pCR template更加靈活A(yù)llows choice of primerscDNA from a single RT can be used in several PCRsWide choi

19、ce of RT and PCR enzymes減少反應(yīng)時(shí)間Fewer pipetting stepsReduced total time of RT-PCR可以最大擴(kuò)增到14 kbAmplify RNA sequences up to 14 kb27Transcriptor HiFi cDNA Synthesis KitFeaturesBlend of Transcriptor enzyme and a proof reading proteinAchieve 7-fold higher fidelity compared to normal reverse transcriptasesOb

20、tain full-length cDNA synthesis in only 10minDetect as low as 10 pg template RNAApplications Sequencing transcriptomesQuantitative RT-PCR applicationsRNA splicing analysisCloning genes of interestGenerating cDNA libraries with large and full-length inserts28Product Comparison Transcriptor Reverse Tr

21、anscriptaseTranscriptor Universal cDNA MasterTranscriptor 1st Strand Synthesis KitTranscriptor High Fidelity cDNA Synth. KitSuperScript III 1st Strand Synthesis SystemSensitivity50pg RNA0.1pg RNA1ug RNA10pg RNA1pg RNAMax frag. length14 kb1 kb14 kb14 kb12kbReaction Time30 min10 min30 min10 min50 minRT Temp.60oC60oC60oC60oC50oCFidelity+/-+/-+/-+n/aMaster mixnoyesnononoUsed forqPCR, RTqPCRqPCR, RTqPCR, RTRT29Protector RNase Inhi