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通过实时聚合酶链反应检测外周小鼠白细胞中微量微嵌合DNA的方法

Methodology for Detecting Trace Amounts of Microchimeric DNA from Peripheral Murine White Blood Cells by Real-Time PCR.

作者信息

Artlett Carol M., Dito C Gennaro, Christner Paul J.

机构信息

Division of Rheumatology, Thomas Jefferson University. 233 S 10th Street, Room 509 B.L.S.B., Philadelphia, PA, 19107. USA.

出版信息

Biol Proced Online. 2003;5:103-107. doi: 10.1251/bpo51. Epub 2003 Apr 7.

DOI:10.1251/bpo51
PMID:12734552
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC153846/
Abstract

Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-k(b) murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 microM primer, a 20-fold increase in the median manufacturer's recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 microg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present.

摘要

实时聚合酶链反应（PCR）方法能够成功定量浓度为100个微嵌合细胞/100,000个宿主细胞的微嵌合细胞群体；然而，对于我们报道的存在于小鼠外周血中浓度低至2/100,000个宿主细胞的微量微嵌合白细胞的DNA定量，该方法并不成功。我们报道了一种使用针对H2-k(b)小鼠组织相容性序列一部分的引物的方法，该引物对C57BL/6J小鼠具有特异性。当在11,000 microM引物存在的情况下使用这些引物时，这是制造商推荐浓度的20倍，该检测方法可以优化到在2.5微克载体BALB/cJ DNA（1/100,000）的背景下检测到34皮克的C57BL/6J DNA。这些条件导致的检测限灵敏度是不存在载体DNA时的一半。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2ec8/153846/e528b6d1eb84/bpo_v5_p103_m51f3lg.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2ec8/153846/a346cb24ed8a/bpo_v5_p103_m51f1lg.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2ec8/153846/9c0ee52586d9/bpo_v5_p103_m51f2lg.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2ec8/153846/e528b6d1eb84/bpo_v5_p103_m51f3lg.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2ec8/153846/a346cb24ed8a/bpo_v5_p103_m51f1lg.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2ec8/153846/9c0ee52586d9/bpo_v5_p103_m51f2lg.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2ec8/153846/e528b6d1eb84/bpo_v5_p103_m51f3lg.jpg

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本文引用的文献

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Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR.使用定量实时聚合酶链反应进行性别不匹配小鼠移植中的嵌合体分析。
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Arthritis Rheum. 2000 Nov;43(11):2598-605. doi: 10.1002/1529-0131(200011)43:11<2598::AID-ANR30>3.0.CO;2-8.
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