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A tissue retrieval and postharvest processing regimen for rodent reproductive tissues compatible with long-term storage on the international space station and postflight biospecimen sharing program.

Gupta V, Holets-Bondar L, Roby KF, Enders G, Tash JS - Biomed Res Int (2015)

Bottom Line: Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging.Postfixation processing was also standardized for safe shipment back to our laboratory.Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Mail Stop 3050, 3901 Rainbow Boulevard, HLSIC 3098, Kansas City, KS 66160, USA.

ABSTRACT
Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

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RNA and protein quality of STS-135 uteri and ovaries stabilized in RNAlater for 10 months at −80°C. (a) STS-135 ground control (G16–G26) ovarian and uterine RNA integrity analysis. Total RNA was extracted and examined for RNA quality; (b) Western blot analysis of ERα and actin in STS-135 mouse uterus. Total cell lysates were prepared and subjected to SDS-PAGE (15 μg/lane). Western blot analysis was performed using the corresponding antibodies to check expression levels of the proteins. Representative Immunoblot (top) and its graphical presentation (bottom). Densitometric intensities of specific protein bands were digitally obtained and normalized to β-actin.
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fig7: RNA and protein quality of STS-135 uteri and ovaries stabilized in RNAlater for 10 months at −80°C. (a) STS-135 ground control (G16–G26) ovarian and uterine RNA integrity analysis. Total RNA was extracted and examined for RNA quality; (b) Western blot analysis of ERα and actin in STS-135 mouse uterus. Total cell lysates were prepared and subjected to SDS-PAGE (15 μg/lane). Western blot analysis was performed using the corresponding antibodies to check expression levels of the proteins. Representative Immunoblot (top) and its graphical presentation (bottom). Densitometric intensities of specific protein bands were digitally obtained and normalized to β-actin.

Mentions: Using ovary and uteri harvested from STS 135 mice, we also determined the effect of long-term (10 months) preservation in RNAlater on RNA and protein quality (Figure 7). This time frame was chosen to mimic a possible storage scenario that could occur on the ISS. Ovaries and uterus from STS-135 ground controls stabilized in RNAlater for 10 months showed excellent RNA quality (Figure 7(a)) and yield in range 6–8 μg.


A tissue retrieval and postharvest processing regimen for rodent reproductive tissues compatible with long-term storage on the international space station and postflight biospecimen sharing program.

Gupta V, Holets-Bondar L, Roby KF, Enders G, Tash JS - Biomed Res Int (2015)

RNA and protein quality of STS-135 uteri and ovaries stabilized in RNAlater for 10 months at −80°C. (a) STS-135 ground control (G16–G26) ovarian and uterine RNA integrity analysis. Total RNA was extracted and examined for RNA quality; (b) Western blot analysis of ERα and actin in STS-135 mouse uterus. Total cell lysates were prepared and subjected to SDS-PAGE (15 μg/lane). Western blot analysis was performed using the corresponding antibodies to check expression levels of the proteins. Representative Immunoblot (top) and its graphical presentation (bottom). Densitometric intensities of specific protein bands were digitally obtained and normalized to β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4309301&req=5

fig7: RNA and protein quality of STS-135 uteri and ovaries stabilized in RNAlater for 10 months at −80°C. (a) STS-135 ground control (G16–G26) ovarian and uterine RNA integrity analysis. Total RNA was extracted and examined for RNA quality; (b) Western blot analysis of ERα and actin in STS-135 mouse uterus. Total cell lysates were prepared and subjected to SDS-PAGE (15 μg/lane). Western blot analysis was performed using the corresponding antibodies to check expression levels of the proteins. Representative Immunoblot (top) and its graphical presentation (bottom). Densitometric intensities of specific protein bands were digitally obtained and normalized to β-actin.
Mentions: Using ovary and uteri harvested from STS 135 mice, we also determined the effect of long-term (10 months) preservation in RNAlater on RNA and protein quality (Figure 7). This time frame was chosen to mimic a possible storage scenario that could occur on the ISS. Ovaries and uterus from STS-135 ground controls stabilized in RNAlater for 10 months showed excellent RNA quality (Figure 7(a)) and yield in range 6–8 μg.

Bottom Line: Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging.Postfixation processing was also standardized for safe shipment back to our laboratory.Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Mail Stop 3050, 3901 Rainbow Boulevard, HLSIC 3098, Kansas City, KS 66160, USA.

ABSTRACT
Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

Show MeSH