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Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues.

Kudo LC, Vi N, Ma Z, Fields T, Avliyakulov NK, Haykinson MJ, Bragin A, Karsten SL - PLoS ONE (2012)

Bottom Line: We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics.High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting.We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

View Article: PubMed Central - PubMed

Affiliation: NeuroInDx, Inc., Signal Hill, California, United States of America. lckudo@neuroindx.com

ABSTRACT
We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

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Total RNA isolated from CTAS microdissected samples shows good integrity. A.Representative quality of total RNA isolated from sucrose treated (1–4) and fresh frozen (5–6) mouse (1–2, 5–6) and rat (3–4) brain tissue samples. RNA integrity numbers (RIN) are shown for each sample. B. Mean RNA integrity numbers (RIN; n≥3) over dissection time show slow decline over time but remain within acceptable range for further analysis. Only 120–130 minutes time point demonstrate significant difference from RNA isolated immediately. C. Representative quality of total RNA isolated from brain tissues (mouse cortex) at different time points. D. Representative RNA isolated from liver and kidney tissues using CTAS based procedure demonstrates acceptable RNA quality after 30 minutes (liver) and 60 minutes (kidney) of microdissection.
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pone-0041564-g007: Total RNA isolated from CTAS microdissected samples shows good integrity. A.Representative quality of total RNA isolated from sucrose treated (1–4) and fresh frozen (5–6) mouse (1–2, 5–6) and rat (3–4) brain tissue samples. RNA integrity numbers (RIN) are shown for each sample. B. Mean RNA integrity numbers (RIN; n≥3) over dissection time show slow decline over time but remain within acceptable range for further analysis. Only 120–130 minutes time point demonstrate significant difference from RNA isolated immediately. C. Representative quality of total RNA isolated from brain tissues (mouse cortex) at different time points. D. Representative RNA isolated from liver and kidney tissues using CTAS based procedure demonstrates acceptable RNA quality after 30 minutes (liver) and 60 minutes (kidney) of microdissection.

Mentions: One of the main concerns for all microdissection techniques is the integrity of RNA molecules used for downstream comparative analyses such as real time PCR or DNA microarrays. To verify RNA quality, total RNA was extracted from CTAS-collected tissue samples acquired from sucrose treated and fresh-frozen mouse brain tissues, as well as fresh frozen mouse liver and kidney (Fig. 7). Independent of tissue preparation method, brain samples collected within 0.5 to 2.0 hours consistently demonstrated high RNA integrity with RIN above 7.5 when evaluated using Agilent Bioanalyzer 2100 (Fig. 7B–C). Kidney samples also showed consistently high quality RNA with high RIN values within 1 hour, and liver samples had expectedly slightly lower RIN values above 6.5 within 30 minutes (Fig. 7D). Total RNA yield from CTAS collected samples was in the expected range of 1.0 to 2.0 µg from 1 mg of wet brain tissue for either Trizol (Life Technologies) or RNeasy kit (Qiagen). To investigate the quality and integrity of protein samples dissected using CTAS, we extracted proteins from the fresh frozen dentate gyrus (DG) and CA2–CA3 tissue areas of adult mouse brain. Proteins extracted from the DG area were labeled with Cy3 and proteins from CA3 areas Cy5 dyes, correspondingly, mixed together and analyzed using 2D gel electrophoresis (Fig. 8). After electrophoresis and scanning on the Typhoon, fluorescently labeled protein images were analyzed using differential in-gel analysis (DIA) module of DeCyder software, which detected 1,613 spots corresponding to individual protein species resolved on the gel. According to DIA, the majority of proteins (97%) in both CA3 and DG samples have the same relative abundances in both tissue types. We also observed differential protein abundances for several protein spots. Some of the differentially expressed proteins were picked from the gel and identified using mass spectrometry (MS) (Fig. 8). Protein identification was performed using the Peptide Mass Fingerprinting (PMF) method and TOF/TOF sequencing of peptides. The PMF results showed that observed peptides span the whole length of the analyzed proteins, pointing to the maintenance of their integrity (1). Several examples are HS90B (23% coverage, 12 matched peptides), CALB2 (32%, 7 peptides), SPEE (26%, 7peptides) and PP2AB (36%, 8 peptides) (Dataset S1). Three identified proteins enriched in CA2–CA3 area corresponded to Calretinin (Calb2; 1.8 fold), Neurocalcin-delta (Ncald, 1.8 fold) and Profilin-1 (Pfn1; 1.65 fold). All three of the identified proteins were previously shown to have higher expression levels in the stratum oriens and pyramidal layer of CA2–CA3 areas compared to the dentate gyrus in line with our findings (Allen Mouse Brain Atlas; [34], [35]). MS analysis has shown that molecular weights and isoelectric points (pI) of all identified proteins accurately matched their position on the gel. We did not detect any evidence of protein degradation; hence we concluded that the quality of the samples isolated by CTAS from tissue sections is sufficient for proteomics studies.


Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues.

Kudo LC, Vi N, Ma Z, Fields T, Avliyakulov NK, Haykinson MJ, Bragin A, Karsten SL - PLoS ONE (2012)

Total RNA isolated from CTAS microdissected samples shows good integrity. A.Representative quality of total RNA isolated from sucrose treated (1–4) and fresh frozen (5–6) mouse (1–2, 5–6) and rat (3–4) brain tissue samples. RNA integrity numbers (RIN) are shown for each sample. B. Mean RNA integrity numbers (RIN; n≥3) over dissection time show slow decline over time but remain within acceptable range for further analysis. Only 120–130 minutes time point demonstrate significant difference from RNA isolated immediately. C. Representative quality of total RNA isolated from brain tissues (mouse cortex) at different time points. D. Representative RNA isolated from liver and kidney tissues using CTAS based procedure demonstrates acceptable RNA quality after 30 minutes (liver) and 60 minutes (kidney) of microdissection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404047&req=5

pone-0041564-g007: Total RNA isolated from CTAS microdissected samples shows good integrity. A.Representative quality of total RNA isolated from sucrose treated (1–4) and fresh frozen (5–6) mouse (1–2, 5–6) and rat (3–4) brain tissue samples. RNA integrity numbers (RIN) are shown for each sample. B. Mean RNA integrity numbers (RIN; n≥3) over dissection time show slow decline over time but remain within acceptable range for further analysis. Only 120–130 minutes time point demonstrate significant difference from RNA isolated immediately. C. Representative quality of total RNA isolated from brain tissues (mouse cortex) at different time points. D. Representative RNA isolated from liver and kidney tissues using CTAS based procedure demonstrates acceptable RNA quality after 30 minutes (liver) and 60 minutes (kidney) of microdissection.
Mentions: One of the main concerns for all microdissection techniques is the integrity of RNA molecules used for downstream comparative analyses such as real time PCR or DNA microarrays. To verify RNA quality, total RNA was extracted from CTAS-collected tissue samples acquired from sucrose treated and fresh-frozen mouse brain tissues, as well as fresh frozen mouse liver and kidney (Fig. 7). Independent of tissue preparation method, brain samples collected within 0.5 to 2.0 hours consistently demonstrated high RNA integrity with RIN above 7.5 when evaluated using Agilent Bioanalyzer 2100 (Fig. 7B–C). Kidney samples also showed consistently high quality RNA with high RIN values within 1 hour, and liver samples had expectedly slightly lower RIN values above 6.5 within 30 minutes (Fig. 7D). Total RNA yield from CTAS collected samples was in the expected range of 1.0 to 2.0 µg from 1 mg of wet brain tissue for either Trizol (Life Technologies) or RNeasy kit (Qiagen). To investigate the quality and integrity of protein samples dissected using CTAS, we extracted proteins from the fresh frozen dentate gyrus (DG) and CA2–CA3 tissue areas of adult mouse brain. Proteins extracted from the DG area were labeled with Cy3 and proteins from CA3 areas Cy5 dyes, correspondingly, mixed together and analyzed using 2D gel electrophoresis (Fig. 8). After electrophoresis and scanning on the Typhoon, fluorescently labeled protein images were analyzed using differential in-gel analysis (DIA) module of DeCyder software, which detected 1,613 spots corresponding to individual protein species resolved on the gel. According to DIA, the majority of proteins (97%) in both CA3 and DG samples have the same relative abundances in both tissue types. We also observed differential protein abundances for several protein spots. Some of the differentially expressed proteins were picked from the gel and identified using mass spectrometry (MS) (Fig. 8). Protein identification was performed using the Peptide Mass Fingerprinting (PMF) method and TOF/TOF sequencing of peptides. The PMF results showed that observed peptides span the whole length of the analyzed proteins, pointing to the maintenance of their integrity (1). Several examples are HS90B (23% coverage, 12 matched peptides), CALB2 (32%, 7 peptides), SPEE (26%, 7peptides) and PP2AB (36%, 8 peptides) (Dataset S1). Three identified proteins enriched in CA2–CA3 area corresponded to Calretinin (Calb2; 1.8 fold), Neurocalcin-delta (Ncald, 1.8 fold) and Profilin-1 (Pfn1; 1.65 fold). All three of the identified proteins were previously shown to have higher expression levels in the stratum oriens and pyramidal layer of CA2–CA3 areas compared to the dentate gyrus in line with our findings (Allen Mouse Brain Atlas; [34], [35]). MS analysis has shown that molecular weights and isoelectric points (pI) of all identified proteins accurately matched their position on the gel. We did not detect any evidence of protein degradation; hence we concluded that the quality of the samples isolated by CTAS from tissue sections is sufficient for proteomics studies.

Bottom Line: We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics.High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting.We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

View Article: PubMed Central - PubMed

Affiliation: NeuroInDx, Inc., Signal Hill, California, United States of America. lckudo@neuroindx.com

ABSTRACT
We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

Show MeSH
Related in: MedlinePlus