Dataset Information

Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues.

ABSTRACT:

Background

Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues.

Results

Here we applied for the first time WGA on laser micro-dissected single cells from stained tissue sections, and developed a protocol for sequentially performing the two procedures. The combined procedure allows correlating the cell's genome with its natural morphology and precise anatomical position. From each cell we amplified 122 genomic and mitochondrial loci. In cells obtained from fresh tissue sections, 64.5% of alleles successfully amplified to approximately 700000 copies each, and mitochondrial DNA was amplified successfully in all cells. Multiplex PCR amplification and analysis of cells from pre-stored sections yielded significantly poorer results. Sequencing and capillary electrophoresis of WGA products allowed detection of slippage mutations in microsatellites (MS), and point mutations in P53.

Conclusion

Comprehensive genomic analysis of single cells from stained tissue sections opens new research opportunities for cell lineage and depth analyses, genome-wide mutation surveys, and other single cell assays.

SUBMITTER: Frumkin D

PROVIDER: S-EPMC2266725 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Publications

Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues.

Frumkin Dan D Wasserstrom Adam A Itzkovitz Shalev S Harmelin Alon A Rechavi Gideon G Shapiro Ehud E

BMC biotechnology 20080220

<h4>Background</h4>Whole genome amplification (WGA) and laser assisted micro-dissection represent two recently developed technologies that can greatly advance biological and medical research. WGA allows the analysis of multiple genomic loci from a single genome and has been performed on single cells from cell suspensions and from enzymatically-digested tissues. Laser micro-dissection makes it possible to isolate specific single cells from heterogeneous tissues.<h4>Results</h4>Here we applied for ...[more]

PMID: 18284708

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Project description:BackgroundThe methods used for sample selection and processing can have a strong influence on the expression values obtained through microarray profiling. Laser capture microdissection (LCM) provides higher specificity in the selection of target cells compared to traditional bulk tissue selection methods, but at an increased processing cost. The benefit gained from the higher tissue specificity realized through LCM sampling is evaluated in this study through a comparison of microarray expression profiles obtained from same-samples using bulk and LCM processing.MethodsExpression data from ten lung adenocarcinoma samples and six adjacent normal samples were acquired using LCM and bulk sampling methods. Expression values were evaluated for correlation between sample processing methods, as well as for bias introduced by the additional linear amplification required for LCM sample profiling.ResultsThe direct comparison of expression values obtained from the bulk and LCM sampled datasets reveals a large number of probesets with significantly varied expression. Many of these variations were shown to be related to bias arising from the process of linear amplification, which is required for LCM sample preparation. A comparison of differentially expressed genes (cancer vs. normal) selected in the bulk and LCM datasets also showed substantial differences. There were more than twice as many down-regulated probesets identified in the LCM data than identified in the bulk data. Controlling for the previously identified amplification bias did not have a substantial impact on the differences identified in the differentially expressed probesets found in the bulk and LCM samples.ConclusionLCM-coupled microarray expression profiling was shown to uniquely identify a large number of differentially expressed probesets not otherwise found using bulk tissue sampling. The information gain realized from the LCM sampling was limited to differential analysis, as the absolute expression values obtained for some probesets using this study's protocol were biased during the second round of amplification. Consequently, LCM may enable investigators to obtain additional information in microarray studies not easily found using bulk tissue samples, but it is of critical importance that potential amplification biases are controlled for.

Project description:Sry is a gene known to be essential for testis determination but is also transcribed in adult male tissues. The laboratory rat, Rattus norvegicus, has multiple Y chromosome copies of Sry while most mammals have only a single copy. DNA sequence comparisons with other rodents with multiple Sry copies are inconsistent in divergence patterns and functionality of the multiple copies. To address hypotheses of divergence, gene conversion and functional constraints, we sequenced Sry loci from a single R. norvegicus Y chromosome from the Spontaneously Hypertensive Rat strain (SHR) and analyzed DNA sequences for homology among copies. Next, to determine whether all copies of Sry are expressed, we developed a modification of the fluorescent marked capillary electrophoresis method to generate three different sized amplification products to identify Sry copies. We applied this fragment analysis method to both genomic DNA and cDNA prepared from mRNA from testis and adrenal gland of adult male rats.Y chromosome fragments were amplified and sequenced using primers that included the entire Sry coding region and flanking sequences. The analysis of these sequences identified six Sry loci on the Y chromosome. These are paralogous copies consistent with a single phylogeny and the divergence between any two copies is less than 2%. All copies have a conserved reading frame and amino acid sequence consistent with function. Fragment analysis of genomic DNA showed close approximations of experimental with predicted values, validating the use of this method to identify proportions of each copy. Using the fragment analysis procedure with cDNA samples showed the Sry copies expressed were significantly different from the genomic distribution (testis p < 0.001, adrenal gland p < 0.001), and the testis and adrenal copy distribution in the transcripts were also significantly different from each other (p < 0.001). Total Sry transcript expression, analyzed by real-time PCR, showed significantly higher levels of Sry in testis than adrenal gland (p, 0.001).The SHR Y chromosome contains at least 6 full length copies of the Sry gene. These copies have a conserved coding region and conserved amino acid sequence. The pattern of divergence is not consistent with gene conversion as the mechanism for this conservation. Expression studies show multiple copies expressed in the adult male testis and adrenal glands, with tissue specific differences in expression patterns. Both the DNA sequence analysis and RNA transcript expression analysis are consistent with more than one copy having function and selection preventing divergence although we have no functional evidence.

Dataset Information

Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues.

Background

Results

Conclusion

Publications

Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues.

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