qPCR试验从设计primer开始到-计算方法详例

1、本人在日本留学虽然对于具体的原理不是特别理解，现把详细的实验real time PCR的实验方法写出来让大家来参考。第一步： primer设计Primer design:1. Find out the mRNA of related gene, for example: Mus musculus transgelin(Tagln) SM22a, mRNA<-> C? 安全 https 7wwncbi.nlm.nih.c|ov/nuccore/NM_011526.5七 NC8【 Resources *1 How To lyJNucleotideNucleotide AdvancedG

2、enQank Mus musculus transgelin (Tagln), mRNANCBI Reference Sequence: NM_O11526.5Go to：日LOCL-S DEFIMTIOX AL'tESSLON YERSIflX KE1WDS S01RCEORGANISMREFERENCEAUTHORS TITLEFASTA Gr 自口 hie一1152615S6 bp mRNA linear ROD 16-0CT-20L7musculus transel in (Taehi mRNA.il_uil52ti 011526.5 R-fS-q. Mus musculus

3、Uious iddus«) 耻5 mu箕ulu$ Ejkaryota; Metazoa： Chordatar Cianiata： Ver：ebraia; Euteleostujii. Haiimalia; Eutheria; EuarchontogI ljes; Glires; Rodentia; 乂加inorpha; Muridae; Miirinae: >his, Yuj 1 (bases 1 to 1586) Aldeiri B, Rt?astalu Uh Albertini A. 'ong _L 如mbit口 A and Cossu G. 'I'

4、ansgelin-expi'essing myofibiLo：ilasts mrcht-strata ventral midlinp ji. 1n +，ai ni rITtn" LTjn. + jn. 二 ax 1 1 anNCBI> Nucleotide> GenBank F I- U,51J UI 51144* 5JLL*iC£, 114tziAM - i nf erence= *al i gnn&ent: 5pl i fin; 1 1 39.父 CDS238,. 843/gene "Ta gin"" /gene

5、_synonym=*Sii22; Sm22a; Ws3i0' /note=*Sjn22aIpha; actin-assaciated protein p27; snooth muscle protein 22-alpha; SM22alphe" /codon_start=l product-," tiansgel in1,/protein_id=飞P 135后孤 I”Mb xif=*CCDS：£rDS23137. TORIGIN1611211S1ggtstctttc cccaaatatg gagcct9tgt sgagtgagtE gggcggcccg g

6、ggtggtgag ccaagcagac ircearggtC 眶监Hggggc gccaecgggc 韶cn酬g自gg Twaicaci gcctaggcgg ccitTaaacc ceicac<cag ccggcgcccc ggcccgtctg ccccagccca 目acacc目皆不耳 ctactcTcct fccflTccac aaacgaccaa gcctTctctg cctraarSBF241 . ， ， ,301II"-'UL ELH - I'-361，”三碇月1牌：以斗：1 t 孔其曰 ggr1£t 炉42J4S1541:ddEHlId

7、tU JHM1L m：Edt：1.EH4：HlE It.LH：二立北1 芯11百打。：1/IgH匕 EE601-1:-|丁:.-:.>6611 1 一，,1，:入，：,.； - ''.J1 L . |,1，'；5 .1 y J'.卜：”721 " &:781Etaca籽aKgag ctccagt?c LEKcaLfiiica 只艮ctalMHg匚 gaccccfiE?ca tcd：cag841面骷狂日 aggccniicce rxagCTKCftg catccTsctt aRcctsccrc acaafirRccT2. open the

8、website of Primer-Blast1. Enter accession, gi, or FASTANCBI Reference Sequnece:NM_011526.5 , rangePrimer ParametersA nefseq mRNA sequence as PCF? template input is required for opPrirner must span an exon-exon junction 丁Exon at 5' side Exon at 3' side74Exon/intron selectionExon junction span

9、E>on junction match2.Intron fidinkmIntron length rangePrimer pair must t>e separate oy " least one intron or the corresponding genomicMinMax100。1000000 MNote: Parameter values that differ fPrimer Pair Specificity Checking ParametersSpecificity checkSearch mtxie* Enable search for primeir

10、pairs specific to the intended PCR lemplte v User glidedt . tDatabaseRefseq mRMA。EKdu$k>nExclude prtdicu Rvfseq transcripts (accosfior with XM. XR prefix ExducOrganismHome 5号 ptensEntrez query ：i. di.ir. i)Enter an organism name) (or crg«riirsm group n3me sucn as enterobaeleriaceaie. rodent!

11、Add MQ整*Primer specificity stringencyMax target &izeAllow splice variantsPnnu'T must have Jt Wa寸 2 T tot?il mismatches tn unintended trgpts. includ st east 2 < rnrsnstches wilhin th»hsl 5 ，bpatths3dend .Ignarp targets that have 6 ， 0r more mismaiches to the primer ” 4000.* Al lew pni

12、Tier to amplify mRNA splice van 争 nl* (requkrat rw 幅 eq mRMAa 旦 P3, * taiDI 双x总:工松修二.1说一，；：心大洸0。二吟:；g面叮大心'虫三' ?mWrite the organism that you use for mice for homo sapiens,mice: Mus musculus (taxid:10090)95。PrimerPair40.0070.00(For thermodynamic alignment rAny3,45.035.0_(For thermodynamic alig

13、nment rAny3*45.035.0(For thermodynamic alignment r240(For thermodynamic alignment model only)PrimerPair120024.00(For old secondary structure aligAny3，8003 00(For old secondary structure aligAny3,8003 00(For old secondary structure aligUse Thermodynamic Oligo Alignment Use ThermodynamicVldllipMax Pol

14、y-XMax T StabilityMax GC in primer 31 endSecondary StructureAlignment MethodsTH: Max Template MisprimingTH: Max SelfComplementarityTH: Max PairComplementarityTH: Max Primer HairpinMax Template MisprimingMax Self ComplementarityMax Pair ComplementarityExcluded regionsOverlap junctions5, side overlaps

15、 3' side overlaps7Overlap junctionsConcentration of monovalent cationsConcentration of divalent cationsConcentration of dNTPsSalt correction formulaTable of thermodynamic parameters Annealing Oligo ConcentrationSNP handlingRepeat filterLow complexity filter5, side overlaps 3* side overlaps74Mini

16、mal number of nucleotides that the left or the right primer miSantaLucia 1998“SantaLucia 1998 050.09Pnmer binding site may not contain known SNP oAutomatic “Avoid repeat region tor pnmer selection by filtering with repeat d . Avoid low complexity region for primer selectionInternal hybridization oli

17、go parametersHybridization oligoRick internal hybndizatlon oligoMinOptMaxHyb Oligo Size182027MinOptMaxHyb Oligo tm57.060063.0MlnOptMaxHyb Oligo GC%20.0|5080.0Get Primers(06 安全 /tools/primer-blast/primertool.cgiPrimer-BLASTPrimer designing tool NCBI/ Primer-BLAST: Making pr

18、imers specific to your PCR template. more .StatusCurrent timeSubmitted27 October 2017,06:30:29CheckPrimer pair 1Sequence (5'->3')Forward primerGCIIIGGGCAGIIIGGCIGIReverse primerTCTTATGCTCCTGGGCIIICTTCAProduct length88Exon junction698/699 (reverse primer) on templatePlus2063165062.0355.003

19、.00Minus2471869561.8645.833.00Template strandLength Start Stop Tm GC% SelfNM 011526.53.将设计的primer序列交给公司合成就可以了提取mRNA1 .对于组织，如血管。在冷的PBS液体中取出血管后马上放入液氮中。带全部样品提取完后，用粉碎机粉碎。大概20次/秒速度，10秒每次，3次。2 .加入1ml Trizon,常温静止30min3 .放于冰上， 加入 200ul chloroform ,震荡混匀。13000rpm, 4C,离心 10-15min4 .取上清液，大概500-600ul5 .加入 500ul

20、2-propronaol ,震荡混匀，13000rpm, 4C, 离心 10-15min 。（因小 鼠组织太小，加入了 4ul Dr.gene, precipitation ）6 .去掉液体，加入80%酒精500ul（一定要用无核酸酶的水，PCR用水），离心13000rpm, 4C, 10-15min ,重复操作 2-3 次。7 .去液体，干燥，加入PCR用水，溶解mRNA8 .测量RNA含量。9 .定量RNA含量，保证样品中有 650ng/ul10 . RT-RCR5*buffer 4ul dNTP 8ulRadom primer 1ulRTace0.5ulRPI0.5ulmRNA:6ul30C, 10min ; 42 C, 60min ; 99 C, 5min ; 4C, 8完成后稀释样品5倍保存待用18s做内参在保存用样品的基础上再稀释50倍11 . Real-time PCR每个样品做1次重复SYBR qPCR mix: 7.5ulROX:0.3ulPrimer F: 0.5025ulPrimer R: 0.5025ulDW 4.695cDNA:1.75HOT start: 1min ,