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Angiotensinogen RT-PCR protocol

Angiotensinogen RT-PCR protocol - Real-Time PCR and Quantitative PCR Forum

Angiotensinogen RT-PCR protocol - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 06-19-2006, 04:31 AM
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Default Re: Expected band for PCR smears..



Could u please tell me about the Angiotensinogen RT-PCR protocol
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Old 06-19-2006, 06:49 PM
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Default Re: Angiotensinogen RT-PCR protocol

I found this RT-PCR protocol:

Reverse-transcription PCR (RT-PCR)
RT-PCR for Angiotensinogen

First-strand cDNA was generated from 2.5 µg denatured total RNA with 20 U avian myeloblastosis virus reverse transcriptase, 1 µg random hexamers and reagents from Promega Corp. (50 min at 42 C and 4 min at 72 C). PCR primers, specific for renin (23), ACE (24, 25), cathepsin D (26), and cathepsin G (27) cDNA, were designed to span at least one intron. PCR was performed in 1x PCR buffer with 0.4 mmol/L deoxynucleotide triphosphate (Boehringer Mannheim, Mannheim, Germany), 1 µmol/L each of sense and antisense primers (Table 1), 2.5 U Taq polymerase (Boehringer Mannheim), and 2.5 U Pfu (Stratagene, La Jolla, CA), in a final vol of 50 µL, using a GeneAmpPCR system 9600 (Perkin-Elmer Corp., Norwalk, CT). Cycle conditions are available on request. Control samples for detection of possible PCR contamination were included in all RT-PCR experiments and were routinely negative.

Primers
Primer Sequence (5' to 3') Nucleotide position

hu Ang F TCC ACC TCG TCA TCC ACA 122–139

hu Ang R GGC TCC CAG ATA GAG AGA 433–450


Human Adipose Tissue Expresses Angiotensinogen and Enzymes Required for Its Conversion to Angiotensin II1

Cecilia Karlsson, Kajsa Lindell, Malin Ottosson, Lars Sjöström, Björn Carlsson and Lena M. S. Carlsson
The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 11 3925-3929

Last edited by admin; 06-19-2006 at 07:12 PM.
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Old 06-19-2006, 06:50 PM
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Default Re: Angiotensinogen RT-PCR protocol

Do a Pubmed Search for Angiotensinogen.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed
I am not sure what model (human, or mouse etc) you are using.

Last edited by admin; 06-19-2006 at 07:12 PM.
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