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A PCR/restriction digestion assay for the detection of the transcript variants 1 and 2 of POU5F1.

Panagopoulos, Ioannis LU ; Möller, Emely LU ; Isaksson, Margareth LU and Mertens, Fredrik LU (2008) In Genes, Chromosomes and Cancer 47. p.521-529
Abstract
POU5F1 has two alternatively spliced transcripts: a long (variant 1, NM_002701) and a short (variant 2, NM_203289) transcript. Only variant 1 is a key regulator of pluripotency. Hence, it is important to be able to distinguish this transcript from variant 2 and from the many pseudogenes present in the genome. Previous studies on the expression of POU5F1 were, however, usually carried out without considering the existence of the two transcripts and the pseudogenes which could be the source of false positive RT-PCR amplification. Here, we establish an RT-PCR/restriction digestion analysis to distinguish variant 1 of POU5F1 from variant 2 and all its currently known pseudogenes. Variant 1 has ApaI and Tsp45I restriction sites, which are not... (More)
POU5F1 has two alternatively spliced transcripts: a long (variant 1, NM_002701) and a short (variant 2, NM_203289) transcript. Only variant 1 is a key regulator of pluripotency. Hence, it is important to be able to distinguish this transcript from variant 2 and from the many pseudogenes present in the genome. Previous studies on the expression of POU5F1 were, however, usually carried out without considering the existence of the two transcripts and the pseudogenes which could be the source of false positive RT-PCR amplification. Here, we establish an RT-PCR/restriction digestion analysis to distinguish variant 1 of POU5F1 from variant 2 and all its currently known pseudogenes. Variant 1 has ApaI and Tsp45I restriction sites, which are not present in the pseudogenes or in variant 2. Thus, ApaI- and Tsp45I- digestions of POU5F1 PCR fragment, amplified with primers flanking these sites, are sufficient to identify the true variant 1 of POU5F1. To study the expression of variant 2 of POU5F1, two forward primers in the 5'-region that are not present in variant 1 were combined with reverse primers located in exon 3 of POU5F1 common to both transcripts. The assay was applied on 10 samples from peripheral blood leukocytes and commercially available ready-cDNAs from leukocytes and testis. We found that only variant 2 was expressed in leukocytes and testis and that the extracted RNA was not completely DNA free, despite DNAse treatment. This trace amount of DNA is a source of false positive RT-PCR amplifications. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. (c) 2008 Wiley-Liss, Inc. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
in
Genes, Chromosomes and Cancer
volume
47
pages
521 - 529
publisher
John Wiley & Sons
external identifiers
  • pmid:18335506
  • wos:000255386700008
  • scopus:43049154741
ISSN
1045-2257
DOI
10.1002/gcc.20555
language
English
LU publication?
yes
id
d60ab95b-3fdf-4be3-aab5-1e8d25762b4c (old id 1052543)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/18335506?dopt=Abstract
date added to LUP
2008-04-02 16:36:43
date last changed
2017-01-01 07:32:16
@article{d60ab95b-3fdf-4be3-aab5-1e8d25762b4c,
  abstract     = {POU5F1 has two alternatively spliced transcripts: a long (variant 1, NM_002701) and a short (variant 2, NM_203289) transcript. Only variant 1 is a key regulator of pluripotency. Hence, it is important to be able to distinguish this transcript from variant 2 and from the many pseudogenes present in the genome. Previous studies on the expression of POU5F1 were, however, usually carried out without considering the existence of the two transcripts and the pseudogenes which could be the source of false positive RT-PCR amplification. Here, we establish an RT-PCR/restriction digestion analysis to distinguish variant 1 of POU5F1 from variant 2 and all its currently known pseudogenes. Variant 1 has ApaI and Tsp45I restriction sites, which are not present in the pseudogenes or in variant 2. Thus, ApaI- and Tsp45I- digestions of POU5F1 PCR fragment, amplified with primers flanking these sites, are sufficient to identify the true variant 1 of POU5F1. To study the expression of variant 2 of POU5F1, two forward primers in the 5'-region that are not present in variant 1 were combined with reverse primers located in exon 3 of POU5F1 common to both transcripts. The assay was applied on 10 samples from peripheral blood leukocytes and commercially available ready-cDNAs from leukocytes and testis. We found that only variant 2 was expressed in leukocytes and testis and that the extracted RNA was not completely DNA free, despite DNAse treatment. This trace amount of DNA is a source of false positive RT-PCR amplifications. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. (c) 2008 Wiley-Liss, Inc.},
  author       = {Panagopoulos, Ioannis and Möller, Emely and Isaksson, Margareth and Mertens, Fredrik},
  issn         = {1045-2257},
  language     = {eng},
  pages        = {521--529},
  publisher    = {John Wiley & Sons},
  series       = {Genes, Chromosomes and Cancer},
  title        = {A PCR/restriction digestion assay for the detection of the transcript variants 1 and 2 of POU5F1.},
  url          = {http://dx.doi.org/10.1002/gcc.20555},
  volume       = {47},
  year         = {2008},
}