【求助】知道Realtime PCR引物，怎么确定RT-PCR产物长度？

最新回复

• bring (2016-4-08 15:05:50)

  
  第三个问题：把图中的tcatagacacagaaacagctttgaaa;ctccagggcgcattcct放到Nucleotide blast中有4个大鼠的，为什么bp值都不一样啊，怎么选择呢？（见下图）

  
  99162057.jpg

• bring (2016-4-08 15:06:21)

  
  图中的bp值都不一样，试验中怎么选择啊？

  
  39814881.jpg

• 131415 (2016-4-08 15:06:39)

  
  去Pubmed找到大鼠11β-HSD1序列，引物中间的大小（含引物）就是你的产物大小。
  real time产物一般在２００以内。
  你也可以自己设计引物

• 131415 (2016-4-08 15:07:23)

  那个bp值不是产物大小。
  选一个出来查对引物或设计引物都可以

• bring (2016-4-08 15:08:12)

  QUOTE:

  原帖由 131415 于 2016-4-8 15:06 发表 bbcodeurl('http://bbs.antpedia.com/images/common/back.gif', '')
  
  去Pubmed找到大鼠11β-HSD1序列，引物中间的大小（含引物）就是你的产物大小。
  real time产物一般在２００以内。
  你也可以自己设计引物

  非常感谢你的指点，但“引物中间的大小（含引物）就是你的产物大小”这句话我不明白，我选了第一个大鼠的序列，粘贴了后面的描述文字，这里面那个是指产物大小啊？
  NCBI Reference Sequence: NM_017080.2
  Rattus norvegicus hydroxysteroid 11-beta dehydrogenase 1 (Hsd11b1), mRNA
  CommentFeaturesSequence
  LOCUS NM_017080 1585 bp mRNA linear ROD 12-APR-2009
  DEFINITION Rattus norvegicus hydroxysteroid 11-beta dehydrogenase 1 (Hsd11b1),
  mRNA.
  ACCESSION NM_017080
  VERSION NM_017080.2 GI:78214364
  KEYWORDS .
  SOURCE Rattus norvegicus (Norway rat)
  ORGANISM Rattus norvegicus
  Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
  Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia;
  Sciurognathi; Muroidea; Muridae; Murinae; Rattus.
  REFERENCE 1 (bases 1 to 1585)
  AUTHORS Liu,Y., Mladinov,D., Pietrusz,J.L., Usa,K. and Liang,M.
  TITLE Glucocorticoid response elements and 11 beta-hydroxysteroid
  dehydrogenases in the regulation of endothelial nitric oxide
  synthase expression
  JOURNAL Cardiovasc. Res. 81 (1), 140-147 (2009)
  PUBMED 18716005
  REMARK GeneRIF: Endogenous 11 beta-HSD1 and 11 beta-HSD2 play significant
  and distinct roles in modulating the effect of glucocorticoids on
  eNOS expression.
  REFERENCE 2 (bases 1 to 1585)
  AUTHORS Liu,Y., Singh,R.J., Usa,K., Netzel,B.C. and Liang,M.
  TITLE Renal medullary 11 beta-hydroxysteroid dehydrogenase type 1 in Dahl
  salt-sensitive hypertension
  JOURNAL Physiol. Genomics 36 (1), 52-58 (2008)
  PUBMED 18826995
  REMARK GeneRIF: suppression of 11 beta-hydroxysteroid dehydrogenase type 1
  expression in the renal medulla attenuates salt-induced
  hypertension in Dahl salt-sensitive rats.
  REFERENCE 3 (bases 1 to 1585)
  AUTHORS Czegle,I., Margittai,E., Senesi,S., Benedetti,A. and Banhegyi,G.
  TITLE Different expression and distribution of 11beta-hydroxysteroid
  dehydrogenase type 1 in obese and lean animal models of type 2
  diabetes
  JOURNAL Acta Physiol Hung 95 (4), 419-424 (2008)
  PUBMED 19009916
  REMARK GeneRIF: prereceptorial glucocorticoid activation is different in
  the liver and adipose tissue of the two diabetes rat models might
  be responsible for the obese and lean phenotypes in type 2 diabetes
  REFERENCE 4 (bases 1 to 1585)
  AUTHORS McCormick,K.L., Wang,X. and Mick,G.J.
  TITLE Modification of microsomal 11beta-HSD1 activity by cytosolic
  compounds: glutathione and hexose phosphoesters
  JOURNAL J. Steroid Biochem. Mol. Biol. 111 (1-2), 18-23 (2008)
  PUBMED 18550363
  REMARK GeneRIF: Modification of microsomal Hsd11b1 activity by cytosolic
  compounds: glutathione and hexose phosphoesters is reported.
  REFERENCE 5 (bases 1 to 1585)

• bring (2016-4-08 15:08:39)

  AUTHORS Paulsen,S.K., Nielsen,M.P., Richelsen,B., Bruun,J.M., Flyvbjerg,A.
  and Pedersen,S.B.
  TITLE Upregulation of adipose 11-beta-hydroxysteroid dehydrogenase type 1
  expression in ovariectomized rats is due to obesity rather than
  lack of estrogen
  JOURNAL Obesity (Silver Spring) 16 (4), 731-735 (2008)
  PUBMED 18379559
  REMARK GeneRIF: The upregulation of 11beta-HSD1 in ovariectomized rats was
  most likely due to changes in body composition rather than lack of
  estrogen
  REFERENCE 6 (bases 1 to 1585)
  AUTHORS Obeid,J. and White,P.C.
  TITLE Tyr-179 and Lys-183 are essential for enzymatic activity of 11
  beta-hydroxysteroid dehydrogenase
  JOURNAL Biochem. Biophys. Res. Commun. 188 (1), 222-227 (1992)
  PUBMED 1417845
  REFERENCE 7 (bases 1 to 1585)
  AUTHORS Moisan,M.P., Edwards,C.R. and Seckl,J.R.
  TITLE Differential promoter usage by the rat 11 beta-hydroxysteroid
  dehydrogenase gene
  JOURNAL Mol. Endocrinol. 6 (7), 1082-1087 (1992)
  PUBMED 1508221
  REFERENCE 8 (bases 1 to 1585)
  AUTHORS Krozowski,Z., Obeyesekere,V., Smith,R. and Mercer,W.
  TITLE Tissue-specific expression of an 11 beta-hydroxysteroid
  dehydrogenase with a truncated N-terminal domain. A potential
  mechanism for differential intracellular localization within
  mineralocorticoid target cells
  JOURNAL J. Biol. Chem. 267 (4), 2569-2574 (1992)
  PUBMED 1733955
  REFERENCE 9 (bases 1 to 1585)
  AUTHORS Agarwal,A.K., Monder,C., Eckstein,B. and White,P.C.
  TITLE Cloning and expression of rat cDNA encoding corticosteroid 11
  beta-dehydrogenase
  JOURNAL J. Biol. Chem. 264 (32), 18939-18943 (1989)
  PUBMED 2808402
  REFERENCE 10 (bases 1 to 1585)
  AUTHORS Monder,C., Shackleton,C.H., Bradlow,H.L., New,M.I., Stoner,E.,
  Iohan,F. and Lakshmi,V.
  TITLE The syndrome of apparent mineralocorticoid excess: its association
  with 11 beta-dehydrogenase and 5 beta-reductase deficiency and some
  consequences for corticosteroid metabolism
  JOURNAL J. Clin. Endocrinol. Metab. 63 (3), 550-557 (1986)
  PUBMED 3460996
  COMMENT VALIDATED REFSEQ: This record has undergone validation or
  preliminary review. The reference sequence was derived from
  CK474613.1 and BC078865.1.
  On Oct 28, 2005 this sequence version replaced gi:8393569.
  Summary: catalyzes the interconversion of cortisol and cortisone;
  plays a role in glucocorticoid metabolism; may regulate blood
  pressure [RGD].
  Publication Note: This RefSeq record includes a subset of the
  publications that are available for this gene. Please see the
  Entrez Gene record to access additional publications.
  PRIMARY REFSEQ_SPAN PRIMARY_IDENTIFIER PRIMARY_SPAN COMP
  1-179 CK474613.1 18-196
  180-1585 BC078865.1 1-1406
  FEATURES Location/Qualifiers
  source 1..1585
  /organism="Rattus norvegicus"
  /mol_type="mRNA"
  /db_xref="taxon:10116"
  /chromosome="13"
  /map="13q27"
  gene 1..1585
  /gene="Hsd11b1"
  /gene_synonym="LRRGT00065"
  /note="hydroxysteroid 11-beta dehydrogenase 1"
  /db_xref="GeneID:25116"
  /db_xref="RATMAP:39283"
  /db_xref="RGD:2834"
  CDS 340..1206
  /gene="Hsd11b1"
  /gene_synonym="LRRGT00065"
  /EC_number="1.1.1.146"
  /note="hydroxysteroid dehydrogenase, 11 beta type 1;
  cortocosteroid 11-beta dehydrogenase"
  /codon_start=1
  /product="hydroxysteroid 11-beta dehydrogenase 1"
  /protein_id="NP_058776.2"
  /db_xref="GI:78214365"
  /db_xref="GeneID:25116"
  /db_xref="RATMAP:39283"
  /db_xref="RGD:2834"
  /translation="MKKYLLPVLVLCLGYYYSTNEEFRPEMLQGKKVIVTGASKGIGR
  EMAYHLSKMGAHVVLTARSEEGLQKVVSRCLELGAASAHYIAGTMEDMAFAERFVVEA
  GKLLGGLDMLILNHITQTTMSLFHDDIHSVRRSMEVNFLSYVVLSTAALPMLKQSNGS
  IAIISSMAGKMTQPLIASYSASKFALDGFFSTIRKEHLMTKVNVSITLCVLGFIDTET
  ALKETSGIILSQAAPKEECALEIIKGTVLRKDEVYYDKSSWTPLLLGNPGRRIMEFLS
  LRSYNRDLFVSN"

• bring (2016-4-08 15:11:50)

  STS 347..482
  /gene="Hsd11b1"
  /gene_synonym="LRRGT00065"
  /standard_name="MARC_17267-17268:1024684507:1"
  /db_xref="UniSTS:268272"
  STS 1203..1328
  /gene="Hsd11b1"
  /gene_synonym="LRRGT00065"
  /standard_name="X83202"
  /db_xref="UniSTS:178555"
  STS 1222..1407
  /gene="Hsd11b1"
  /gene_synonym="LRRGT00065"
  /standard_name="RH128149"
  /db_xref="UniSTS:211457"
  ORIGIN
  1 caaaggatgt ctcggtagga gatgctcagg aacccagccc tgcacagtca tgagcctggc
  61 catctggaag ttgcctctta ctcaatgaaa tggagtaaac attgtccatt atgaaatcca
  121 tcacgcaggc tgccagggac gaatgggatc ccacccaaag ccaatcattg ctctgacagg
  181 gaagttggct agtgctgcct gagactactc cagcctcccc cgtccctgat gtcacaattc
  241 agaggctgct gcctgcctgg gaggttgtag aaagagctgc aggttttctt tgtgtgtcct
  301 acagagctcc ctgagccagg tccctgttgg acggcagtta tgaaaaaata cctcctcccc
  361 gtcctggtgc tctgcctggg ttactactat tctacaaatg aagagttcag accagaaatg
  421 ctccagggga agaaagtgat tgtcactggg gccagcaaag ggatcggaag agaaatggca
  481 tatcatctgt caaaaatggg agcccatgtg gtattgactg caaggtcgga ggaagggctc
  541 cagaaggtgg tgtctcgctg ccttgaactc ggagcagcct ctgctcacta tattgccggc
  601 actatggaag acatggcttt tgcagagcga tttgttgttg aggcaggaaa gctcttgggt
  661 ggactggaca tgctcattct caatcacatc acacagacca ctatgtctct cttccacgat
  721 gatatccact ctgtgcgaag aagcatggag gtcaacttcc tcagctatgt ggtcctgagc
  781 acagcagcct tgcccatgct gaaacagagc aatggcagca ttgccatcat ctcctccatg
  841 gctgggaaaa tgacccaacc tctgattgct tcctactctg caagcaagtt tgctctggat
  901 gggttctttt ccaccattag aaaagaacac ttgatgacca aggtcaacgt gtccatcact
  961 ctctgtgtcc tcggcttcat agacacagaa acagctttga aagaaacctc tgggataatc
  1021 ttgagtcaag ctgctcccaa ggaggaatgc gccctggaga tcatcaaagg cacagttctg
  1081 cgcaaagatg aggtatacta tgacaaatca tcttggactc cacttctgct tgggaatcca
  1141 ggaaggagga tcatggaatt tctttcatta cggtcatata acagggacct atttgtaagc
  1201 aactaggaac tcctgaggcc tggtgaaggg tcttggaaca gtcctgcctg atatttctgt
  1261 gagccctacc cacaaatttc tatgagcccc tttccagaga tacacaaatt ttggggtaca
  1321 cctcaccatg agaaattctt gcaacacttg cacagtaaaa atgtaattgt aataaatgtc
  1381 acaaaccact ttgtagcctg cagttgtgaa cttgattgta actatggatt taagcacaca
  1441 gttgtatcaa ttttacctca tacagaatga aacaatgatg cctaatgtaa cattaaataa
  1501 aataaaggta atatcaactc tataaattca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
  1561 aaaaaaaaaa aaaaaaaaaa aaaaa
  //

• bring (2016-4-08 15:13:02)

  查了一下这个基因只有一个剪切体，我还以为有多个，你不知道选哪一个呢。
  你既然都查到序列了，在序列中找到这两条引物（其中一个引物是反向互补的），中间的不就是你的产物长度吗？
  tcat agacacagaa acagctttga aagaaacctc tgggataatc
  1021 ttgagtcaag ctgctcccaa ggaggaatgc gccctggag
  产物长度是83。