Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of different extraction and storage methods on RNA and protein quality in ovaries, and testes extracted and/or stabilized in TRIzol or RNAlater. (a) Total RNA integrity analysis. Total RNA was isolated from mouse testis and stabilized in TRIzol reagent (RNA yield and quality control (lane 1); RNAlater for 2 wks at 4°C (lane 2); RNAlater for 1 wk at room temperature and 1 wk at 4°C (lane 3); RNAlater for 2 wks at RT (lane 4). (b) Agarose gel electrophoresis of PCR products with primers for IL-1α and GAPDH. For RT PCR total RNA was used after stabilization in TRIzol reagent (lane 1); RNAlater for 2 wks at 4°C (lane 2); RNAlater for 1 wk at RT and 1 wk at 4°C (lane 3); RNAlater for 2 wks at RT (lane 4). (c) Total RNA integrity analysis STS-131 ground (G10, G11, and G12) and flight (F10, F11, and F12) uteri fixed in RNAlater after 30–40 min after euthanasia. (d) Comparison of buffers to remove RNAlater for subsequent western analysis of β-actin integrity in ovaries. Mouse ovaries were stored at RNAlater for 1 wk at RT or 2 wk at 4°C; tissues were homogenized in RIPA lysis buffer (lanes 1, 2) or ProteoJET Lysis reagent (LR) (lanes 3, 4). Total cell lysates were prepared and subjected to SDS-PAGE. Western for β-actin is presented.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4309301&req=5

fig6: Effect of different extraction and storage methods on RNA and protein quality in ovaries, and testes extracted and/or stabilized in TRIzol or RNAlater. (a) Total RNA integrity analysis. Total RNA was isolated from mouse testis and stabilized in TRIzol reagent (RNA yield and quality control (lane 1); RNAlater for 2 wks at 4°C (lane 2); RNAlater for 1 wk at room temperature and 1 wk at 4°C (lane 3); RNAlater for 2 wks at RT (lane 4). (b) Agarose gel electrophoresis of PCR products with primers for IL-1α and GAPDH. For RT PCR total RNA was used after stabilization in TRIzol reagent (lane 1); RNAlater for 2 wks at 4°C (lane 2); RNAlater for 1 wk at RT and 1 wk at 4°C (lane 3); RNAlater for 2 wks at RT (lane 4). (c) Total RNA integrity analysis STS-131 ground (G10, G11, and G12) and flight (F10, F11, and F12) uteri fixed in RNAlater after 30–40 min after euthanasia. (d) Comparison of buffers to remove RNAlater for subsequent western analysis of β-actin integrity in ovaries. Mouse ovaries were stored at RNAlater for 1 wk at RT or 2 wk at 4°C; tissues were homogenized in RIPA lysis buffer (lanes 1, 2) or ProteoJET Lysis reagent (LR) (lanes 3, 4). Total cell lysates were prepared and subjected to SDS-PAGE. Western for β-actin is presented.

Mentions: We compared RNA integrity after preservation testicular tissue in TRIzol reagent and RNAlater (Figure 6(a)). Total RNA integrity analysis demonstrated high quality RNA after storing tissue in RNAlater for 1-2 wk at RT or 4°C compared to TRIzol preservation. Distinct 28S and 18S ribosomal RNA and absence of degraded RNA were observed on the gel. RT-PCR analysis of RNA with primers for GAPDH and IL-1α indicated high quality of expected PCR products in all analyzed samples (Figure 6(b)). Integrity analysis of total RNA isolated from STS-131 ground controls (G10, G11, and G12) and flight (F10, F11, and F12) mouse uteri after 30 to 40 min after euthanasia demonstrate high RNA quality in these samples (Figure 6(c)). We found no differences in RNA quality between all analyzed uteri and ovaries samples placed into RNAlater from 15 min to 40 min after euthanasia.


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)

Effect of different extraction and storage methods on RNA and protein quality in ovaries, and testes extracted and/or stabilized in TRIzol or RNAlater. (a) Total RNA integrity analysis. Total RNA was isolated from mouse testis and stabilized in TRIzol reagent (RNA yield and quality control (lane 1); RNAlater for 2 wks at 4°C (lane 2); RNAlater for 1 wk at room temperature and 1 wk at 4°C (lane 3); RNAlater for 2 wks at RT (lane 4). (b) Agarose gel electrophoresis of PCR products with primers for IL-1α and GAPDH. For RT PCR total RNA was used after stabilization in TRIzol reagent (lane 1); RNAlater for 2 wks at 4°C (lane 2); RNAlater for 1 wk at RT and 1 wk at 4°C (lane 3); RNAlater for 2 wks at RT (lane 4). (c) Total RNA integrity analysis STS-131 ground (G10, G11, and G12) and flight (F10, F11, and F12) uteri fixed in RNAlater after 30–40 min after euthanasia. (d) Comparison of buffers to remove RNAlater for subsequent western analysis of β-actin integrity in ovaries. Mouse ovaries were stored at RNAlater for 1 wk at RT or 2 wk at 4°C; tissues were homogenized in RIPA lysis buffer (lanes 1, 2) or ProteoJET Lysis reagent (LR) (lanes 3, 4). Total cell lysates were prepared and subjected to SDS-PAGE. Western for β-actin is presented.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Effect of different extraction and storage methods on RNA and protein quality in ovaries, and testes extracted and/or stabilized in TRIzol or RNAlater. (a) Total RNA integrity analysis. Total RNA was isolated from mouse testis and stabilized in TRIzol reagent (RNA yield and quality control (lane 1); RNAlater for 2 wks at 4°C (lane 2); RNAlater for 1 wk at room temperature and 1 wk at 4°C (lane 3); RNAlater for 2 wks at RT (lane 4). (b) Agarose gel electrophoresis of PCR products with primers for IL-1α and GAPDH. For RT PCR total RNA was used after stabilization in TRIzol reagent (lane 1); RNAlater for 2 wks at 4°C (lane 2); RNAlater for 1 wk at RT and 1 wk at 4°C (lane 3); RNAlater for 2 wks at RT (lane 4). (c) Total RNA integrity analysis STS-131 ground (G10, G11, and G12) and flight (F10, F11, and F12) uteri fixed in RNAlater after 30–40 min after euthanasia. (d) Comparison of buffers to remove RNAlater for subsequent western analysis of β-actin integrity in ovaries. Mouse ovaries were stored at RNAlater for 1 wk at RT or 2 wk at 4°C; tissues were homogenized in RIPA lysis buffer (lanes 1, 2) or ProteoJET Lysis reagent (LR) (lanes 3, 4). Total cell lysates were prepared and subjected to SDS-PAGE. Western for β-actin is presented.
Mentions: We compared RNA integrity after preservation testicular tissue in TRIzol reagent and RNAlater (Figure 6(a)). Total RNA integrity analysis demonstrated high quality RNA after storing tissue in RNAlater for 1-2 wk at RT or 4°C compared to TRIzol preservation. Distinct 28S and 18S ribosomal RNA and absence of degraded RNA were observed on the gel. RT-PCR analysis of RNA with primers for GAPDH and IL-1α indicated high quality of expected PCR products in all analyzed samples (Figure 6(b)). Integrity analysis of total RNA isolated from STS-131 ground controls (G10, G11, and G12) and flight (F10, F11, and F12) mouse uteri after 30 to 40 min after euthanasia demonstrate high RNA quality in these samples (Figure 6(c)). We found no differences in RNA quality between all analyzed uteri and ovaries samples placed into RNAlater from 15 min to 40 min after euthanasia.

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
Related in: MedlinePlus