Sorting cells for RT-PCR or PCR (latest update)

Recent news on sorting cells for downstream molecular applications

Sorting cells for SNP Analysis:
Clinical scientists at Erasmus Medical Center, Rotterdam have demonstrated an improved diagnostic and prognostic strategy for malignant pleural mesothelioma based on flowcytometry.  They show significant benefits in performance compared to routine cytological examination and the CellSearch platform.

To confirm that a putative malignant disease phenotype was identified, cells from pleural effusions were stained with DRAQ5, anti-CD45 and anti-MCAM.  The different sorted populations were subjected to SNP analysis to determine copy number variation.

Additionally, DRAQ5 was used in a flow cytometric gating strategy for the detection of tumour-associated circulating endothelial cells (CEC).  Presence of tumour-associated CEC appeared to  negatively impact overall survival.

Beije, Nick, et al. "Improved diagnosis and prognostication of patients with pleural malignant mesothelioma using biomarkers in pleural effusions and peripheral blood samples–a short report." Cellular Oncology (2017): 1-9.

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And previously, we reported on..

Cell Cycle-Based PCR:

Solid tumour tissue dissociated cells were sorted based on DRAQ5™ DNA content & S100 phenotype to permit downstream PCR and mutation detection

Douwes Dekker, P. B., et al. "Multiparameter DNA flow‐sorting demonstrates diploidy and SDHD wild‐type gene retention in the sustentacular cell compartment of head and neck paragangliomas: chief cells are the only neoplastic component." 
J Pathol 202.4 (2004): 456-462.

Key finding..
DRAQ5 does not interfere with Taq polymerase

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DNA Content-Based RT-PCR for "RNAseq":

Similarly, RT-PCR has been performed on DRAQ5™ stained neurones. Cells were harvested from fresh GFP mouse hypothalamus tissue digest (papain, DNase) and filtered to recover a single cell suspension.  Cells were sorted directly into lysis buffer (with RNAse inhibitor) in microtitre plate wells for RTase and amplification.

Lam, Brian YH, et al. "Heterogeneity of hypothalamic Pro-opiomelanocortin-expressing neurons revealed by single-cell RNA sequencing." Molecular Metabolism (2017). http://dx.doi.org/10.1016/j.molmet.2017.02.007

Key finding..

DRAQ5™does not interfere with RT'ase or impact on RNA integrity

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Cell Sorting for RNA sequencing: 

Cell sorting for RNA-seq: exclusion of dead/damaged cells (i.e. DRAQ7™+ events) along with high forward scatter was used to select for cardiomyocytes from heart perfusion.  Dead cell exclusion removes risk of unwanted background from fragmented RNA transcripts.

Preissl, S., et al. "Deciphering the Epigenetic Code of Cardiac Myocyte Transcription" 
Circulation Research. 2015; 117: 413-423.
http://circres.ahajournals.org/content/117/5/413.full

The procedure used is described in the supplementary data file:
http://circres.ahajournals.org/content/suppl/2015/06/23/CIRCRESAHA.115.306337.DC1/306337R3_Online.pdf

Key findings ..
DRAQ7™does not interfere with intact cells containing precious, fragile RNA transcripts 

DRAQ7™allows binary exclusion of dead cells - clearly separated from intact cells (stain index difference of >25)

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