【求助】知道Realtime PCR引物，怎么确定RT-PCR产物长度？

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标题:【求助】知道Realtime PCR引物，怎么确定RT-PCR产物长度？

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发表于 2016-4-8 15:04 资料个人空间短消息加为好友

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各位大侠：我的实验中要测大鼠的11β-HSD1这种酶的表达，我查了很多英文文献中都是用real-time pcr测的（见下图中）。但我们的实验室只能做RT-PCR，而文献中用real-time pcr测的只交待了引物，却没说产物长度。我的第一个问题：我打算用文献中real-time pcr法的引物，来放到RT-pcr中去做，可以吗？第二个问题：订引物时不知道产物长度，这种情况怎么办呢？求大侠高人指点迷津，不胜感激！！！

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发表于 2016-4-8 15:05 资料个人空间短消息加为好友

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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）

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发表于 2016-4-8 15:06 资料个人空间短消息加为好友

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图中的bp值都不一样，试验中怎么选择啊？

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发表于 2016-4-8 15:06 资料个人空间短消息加为好友

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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

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发表于 2016-4-8 15:07 资料个人空间短消息加为好友

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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

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发表于 2016-4-8 15:08 资料个人空间短消息加为好友

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QUOTE:

原帖由 131415 于 2016-4-8 15:06 发表 bbcodeurl('http://bbs.antpedia.com/images/common/back.gif', '%s')

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)

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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"

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发表于 2016-4-8 15:11 资料个人空间短消息加为好友

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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
//

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发表于 2016-4-8 15:13 资料个人空间短消息加为好友

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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。