| Autor: |
Vallandingham, J., Peak, A., Morrison, J., Bailey, C., Staehling, K., Perera, A., Zueckert-Gaudenz, K., Kulesa, P., Li, H., Fleharty, B. |
| Jazyk: |
angličtina |
| Rok vydání: |
2014 |
| Předmět: |
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| Popis: |
Laser capture microdissection (LCM) permits the precise isolation of small populations of cells from complex tissue. Without the need for purification, the limited quantities of total RNA in LCM samples can be amplified for global gene expression profiling by RNA-Seq. In this study, two RNA amplification methods were evaluated. As input, 10, 30 and 100 human metastatic melanoma cells (c8161) were harvested by LCM after transplantation into the chick embryonic neural crest microenvironment. For comparison, 10 to 300 cultured c8161 cells were processed in identical manner. The amplified cDNA samples generated with either the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech) or the Ovation RNA-Seq System V2 kit (NuGEN) were fragmented prior to Illumina TruSeq library construction. As references, libraries were prepared from 10 ng of amplified and 1 μg of unamplified total RNA isolated from ∼7 million cultured cells. We show that both kits can be used to perform quantitative transcriptome analysis with as few as 10 cells. The cDNA yields obtained with the NuGEN kit were significantly higher than the Clontech yields. The number of expressed genes (>0 FPKM) increased with higher cell numbers, particularly for the culture-derived samples. Gene length and transcript abundance positively affected correlation with the unamplified reference for both the LCM and culture-derived samples. Considerably different expression profiles were observed for the samples amplified with the NuGEN versus Clontech methods which may be attributed to the different chemistries. Therefore, caution is advised when directly comparing inter-kit data sets. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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