Cell-Specific Molecules Isolation

Capture and isolation of single cells are prerequisites for understanding these cellular variations. However, efficient single cell capture and isolation are complex tasks for any cell population. Therefore, several methods have been introduced to isolate the cell-specific nuclei to avoid the potential concerns of isolation of the whole cell or cell-specific nucleic acids/proteins by labeling them.

TU Tagging

The TU Tagging method can be used for cell-specific gene expression analysis, measurement of RNA synthesis, and degradation rates by isolating newly synthesized RNA from the bulk of cellular RNA.

An analog of the uracil, TU (4-thiouracil), acts on a substrate of UPRT (Uracil Phosphoribosyl Transferase) to subsequently add it into RNA. Simultaneously, this thio-substituted nucleotide acts as a tag-thio-labeled RNA that can be purified using commercially available reagents.

Fig.1 A diagram of the TU Tagging isolation method. (Cleary, 2005)

Advantages of TU Tagging

• No cellular dissociation.
• Suitable for cell-specific RNA isolation.

Limitations of TU Tagging

• Time-consuming for the production of genetic tags.

Poly(A) mRNA Tagging

Poly(A) mRNA tagging was used for profiling gene expression in specific tissues. A poly(A)-binding protein (PABP) fused with a FLAG tag is expressed in particular tissues, total RNA is extracted, and tagged RNA is isolated by immunoprecipitation with a FLAG-tag-specific antibody and amplified. The amplified mRNA is used as a probe in microarray experiments, next-generation sequencing, or for transcriptomic analysis.

Fig.2 A diagram of poly(A) mRNA tagging method. (Roy, 2002)

Advantages of Poly(A) mRNA Tagging

• High Specificity.
• It can isolate the whole RNA effectively.

Limitations of Poly(A) mRNA Tagging

• Antibody-based assay.
• Immunoprecipitation.

Translating Ribosome Affinity Purification (TRAP)

TRAP is used to isolate ribosome-bound mRNA by using the green fluorescent protein (GFP)-tagged ribosomal protein expressed in specific cell types.

Fig.3 A diagram of the TRAP method. (Heiman, 2008)

Advantages of TRAP

• Transcribed mRNA isolation.
• Cost-effective.

Limitations of TRAP

• Genetic tagging.
• It cannot discriminate the RNA from an active or stalled ribosome.
• It cannot investigate noncoding RNA or miRNA.
• High chance of contamination.

miRAP

To analyze miRNA expression in neurons of mice, the miRNA tagging and affinity purification method (miRAP) was established to target specific cell types by the Cre-LoxP binary system. miRAP is based on a miRNA-induced silencing complex in which the Argonaute protein AGO2 binds. miRNA can be purified by antibodies against the engineered epitope-tagged AGO2 (tAGO2) delivered by Cre-LoxP binary expression system to the interest cell.

Fig.4 The pull-down strategy of determining miRNA that targets a specific gene. (Wei, 2014)

Targeted DamID (TaDa)

The TaDa approach enables cell type-specific profiling of chromatin-binding proteins based on DNA adenine methyltransferase identification (DamID).

Fig.5 TaDa can profile the interaction of any protein with DNA or chromatin in a cell-type-specific manner. (Marshall, 2016)

Advantages of TaDa

• Simple, robust.
• No affinity purification.
• No fixation.

Limitations of TaDa

• Time-consuming for the production of genetic tags.

At Creative Biolabs, we can provide TU tagging, poly(A) mRNA tagging, TRAP, miRAP, and TaDa services to isolate molecules from specific single-cell types. If you have any requirements, please feel free to contact us for further communication about your project.

References

1. Cleary, M.D.; et al. Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay. Nature Biotechnology. 2005, 23: 232-237.
2. Roy, P.J.; et al. Chromosomal clustering of muscle-expressed genes in Caenorhabditis elegans. Nature. 2002, 418: 975-979.
3. Heiman, M.; et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell. 2008, 135(4): 738-748.
4. Wei, K.; et al. Affinity purification of binding miRNAs for messenger RNA fused with a common tag. International Journal of Molecular Sciences. 2014, 15(8): 14753-14765.
5. Marshall, O.; et al. Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing. Nature Protocols. 2016, 11: 1586-1598.