Quantification of rat kisspeptin using a novel radioimmunoassay.
Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. How...
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0097611 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849332542723850240 |
|---|---|
| author | James S Kinsey-Jones Kylie E Beale Joy Cuenco Xiao Feng Li Stephen R Bloom Kevin T O'Byrne Kevin G Murphy |
| author_facet | James S Kinsey-Jones Kylie E Beale Joy Cuenco Xiao Feng Li Stephen R Bloom Kevin T O'Byrne Kevin G Murphy |
| author_sort | James S Kinsey-Jones |
| collection | DOAJ |
| description | Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models. |
| format | Article |
| id | doaj-art-8902eb2483424f0ea7f597d29b700ebc |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-8902eb2483424f0ea7f597d29b700ebc2025-08-20T03:46:11ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0195e9761110.1371/journal.pone.0097611Quantification of rat kisspeptin using a novel radioimmunoassay.James S Kinsey-JonesKylie E BealeJoy CuencoXiao Feng LiStephen R BloomKevin T O'ByrneKevin G MurphyKisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models.https://doi.org/10.1371/journal.pone.0097611 |
| spellingShingle | James S Kinsey-Jones Kylie E Beale Joy Cuenco Xiao Feng Li Stephen R Bloom Kevin T O'Byrne Kevin G Murphy Quantification of rat kisspeptin using a novel radioimmunoassay. PLoS ONE |
| title | Quantification of rat kisspeptin using a novel radioimmunoassay. |
| title_full | Quantification of rat kisspeptin using a novel radioimmunoassay. |
| title_fullStr | Quantification of rat kisspeptin using a novel radioimmunoassay. |
| title_full_unstemmed | Quantification of rat kisspeptin using a novel radioimmunoassay. |
| title_short | Quantification of rat kisspeptin using a novel radioimmunoassay. |
| title_sort | quantification of rat kisspeptin using a novel radioimmunoassay |
| url | https://doi.org/10.1371/journal.pone.0097611 |
| work_keys_str_mv | AT jamesskinseyjones quantificationofratkisspeptinusinganovelradioimmunoassay AT kylieebeale quantificationofratkisspeptinusinganovelradioimmunoassay AT joycuenco quantificationofratkisspeptinusinganovelradioimmunoassay AT xiaofengli quantificationofratkisspeptinusinganovelradioimmunoassay AT stephenrbloom quantificationofratkisspeptinusinganovelradioimmunoassay AT kevintobyrne quantificationofratkisspeptinusinganovelradioimmunoassay AT kevingmurphy quantificationofratkisspeptinusinganovelradioimmunoassay |