High-throughput intracellular biopsy of microRNAs for dissecting the temporal dynamics of cellular heterogeneity

Abstract

The capability to analyze small RNAs responsible for post-transcriptional regulation of genes expression is essential for characterizing cellular phenotypes. Here, we describe an intracellular biopsy technique (inCell-Biopsy) for fast, multiplexed, and highly sensitive profiling of microRNAs (miRNAs). The technique uses an array of diamond nanoneedles that are functionalized with size-dependent RNA binding proteins, working as “fishing rods” to directly pull miRNAs out of cytoplasm while keeping the cells alive, thus enabling quasi-single-cell miRNA analysis. Each nanoneedle works as a reaction chamber for parallel in situ amplification, visualization, and quantification of miRNAs as low as femtomolar, which is sufficient to detect miRNAs of a single-copy intracellular abundance with specificity to single-nucleotide variation. Using inCell-Biopsy, we analyze the temporal miRNA transcriptome over the differentiation of embryonic stem cells (ESCs). The combinatorial miRNA expression patterns derived by inCell-Biopsy identify emerging cell subpopulations differentiated from ESCs and reveal the dynamic evolution of cellular heterogeneity.

Single cell multi-miRNAs quantification with hydrogel microbeads for liver cancer cell subtypes discrimination

“…Multi-miRNAs expression analysis in a single cell indicates cellular heterogeneity, and provides a signicant reference for cancer subtypes discrimination. 7,43 Multi-MCPs functionalized hydrogel microbeads were able to amplify multiple miRNAs signals simultaneously in a single microbead, and barely demonstrated nonspecic cross-reactions. HepG2, HCCLM3 and MHCC97L, three liver cancer cell lines, and HHL-5, a normal liver cell line, were chosen as the sample cell lines for liver cell subtypes discrimination.…”

“…Multiplex microRNAs (miR-NAs) regulate the gene network collaboratively to maintain cell renewal, proliferation, and apoptosis. [1][2][3][4][5][6] Different miRNAs expression levels are closely related to cellular heterogeneity, [7][8][9] which plays an important role in the characterization and classication of subpopulations, [10][11][12][13] and contributes to cancer subtypes discrimination. 14,15 However, intracellular multi-miRNAs imaging strategies cannot provide quantitative information.…”

Profiling MicroRNAs with Associated Spatial Dynamics in Acute Tissue Slices

“…To initiate in situ miRNA profiling in acute brain slices, a diamond nanoneedle assisted puncture of cells was performed to temporally disrupt the cell membrane for the delivery of probe sequences to the cells in the tissue (Figure a,b), which is similar to the intracellular delivery procedure as previously demonstrated in cultured cells by our group. ,, Specifically, for the interrogation of miRNA dynamics in a rodent schizophrenia model, rats were repeatedly injected with MK-801, an N -methyl- d -aspartate receptor (NMDAR) antagonist to induce brain impairments mimicking certain aspects of the disease. , Hippocampal slices were isolated from the brain of MK-801-treated schizophrenia (SCZ) or normal control (Norm or N) animals and were placed on a piece of filter paper and covered by medium containing the probe sequences before an intracellular biopsy operation. The probe sequence was designed to be complementary to targeted miRNAs with a short overhang region (Figure c and Supporting Information (SI) Table S1).…”

“…As short noncoding RNAs, miRNAs bind to mRNAs to regulate the post-transcriptional processes of mRNAs, and thereby control gene expressions in a fine-tuning manner. , To date, prevalent techniques to profile the alterations of miRNAs include RNA sequencing (RNA-seq), microarray, and fluorescent in situ hybridization (FISH). ,, To analyze miRNA by sequencing, short RNAs from animal or post-mortem human tissues are isolated by an additional size-fractionation procedure before reverse transcription for further processing; microarray uses immobilized probes to capture miRNAs from cellular lysate by hybridization and further visualizes the results by florescent imaging . Though both RNA-seq and microarray can analyze hundreds of miRNAs for homogenized samples, the techniques do not provide any information regarding the cellular heterogeneity and associated spatial dynamics, and sometime give discrepant results attributed to platforms and workflows. ,− Alternatively, FISH allows in situ hybridization of prelabeled probes with targeted miRNAs in fixed tissues or cells, thus enabling investigation of miRNA spatial distribution but with limited throughput . While barcode-based FISH improves the throughput to hundreds of targets, it only applies to long mRNAs of at least hundreds of nucleotides (NTs) and is not transferrable to miRNAs as short as ∼20 NTs.…”

Perspectives on MicroRNA Study in Oncogenesis: Where Are We?

“…High-throughput technologies based on miR profiling have brought advancements in cancer diagnosis and prognosis [12] . The latest technology for high-throughput miR biopsy, termed inCell-Biopsy, provides a commercially available method for miR profiling in situ in living cells, which could reveal cellular heterogeneity based on miR expression [13] . Moreover, the improvement of conventional miR amplification and detection technologies, such as RT-qPCR, Northern blot, microarray, and RNA-seq, has opened new possibilities for improved clinical diagnosis of cancer patients.…”