Procedure For Isolation Of Nuclei Research Articles

Nuclei isolation of multiple brain cell types for omics interrogation.

We present a nuclei isolation protocol for genomic and epigenomic interrogation of multiple cell type populations in the human and rodent brain. The nuclei isolation protocol allows cell type-specific profiling of neurons, microglia, oligodendrocytes, and astrocytes, being compatible with fresh and frozen samples obtained from either resected or postmortem brain tissue. This 2-day procedure consists of tissue homogenization with fixation, nuclei extraction, and antibody staining followed by fluorescence-activated nuclei sorting (FANS) and does not require specialized skillsets. Cell type-specific nuclei populations can be used for downstream omic-scale sequencing applications with an emphasis on epigenomic interrogation such as histone modifications, transcription factor binding, chromatin accessibility, and chromosome architecture. The nuclei isolation protocol enables translational examination of archived healthy and diseased brain specimens through utilization of existing medical biorepositories.

An efficient procedure for isolation of nuclei from plant protoplasts

The present communication describes an easy, efficient and rapid method for isolation of nuclei from plant protoplasts. Release of nuclei is accomplished by disruption of protoplasts in an appropriate buffer containing a very low concentration (0.01%) of the detergent Triton X-100. The pH of the nuclei isolation buffer (5.3) played a critical role in the recovery of stable nuclei in large numbers. Supplementation of buffer (10 mM MES) with spermine (0.1 mM), dithiothreitol (2.5 mM), ethylenediaminetetraacetic acid (2.5 mM) and Nad and KCl (10 mM each) improved nuclear yield and quality. With the method developed it is possible to routinely recover 95% nuclei from the protoplasts within 30 minutes. The nuclear preparations are of high purity with little detectable cytoplasmic contamination and no clumping of the nuclei. The structural integrity of the nuclei has been assessed and confirmed by Nomarski differential interference contrast optics and ultrastructural observations.

Extraction of rapidly labeled undegraded ribonucleic acid from isolated rat liver nuclei

An important point in all investigations on nuclear RNA is the problem of isolation of pure nuclei. On the other hand, during the isolation of nuclei the nuclear RNA must be preserved from nucleases. In this connection it is not yet clear to what extent the different procedures for isolation of nuclei affect the nuclear RNA. Since there is strong evidence that the RNA is synthesized in the nucleus, all precursors of the cytoplasmic RNA's are expected to be found in the early labeled fractions of the nuclear RNA. It has been shown (1–3) that a RNA fraction having a higher turnover rate is not extractable by the usual phenol procedure. Although it accounts for only a few per cent of the total RNA it is of greatest biological importance, since it comprises both precursors of ribosomal RNA and of messenger RNA (4). The great lability of this RNA is possibly the reason why in different studies varying results were obtained concerning the heterogeneity and the localization of the early labeled RNA isolated from pure nuclei. In our experiments we have studied the electrophoretic and the labeling pattern of the nuclear RNA extracted from pure rat liver nuclei isolated by several different methods. A suitable procedure for isolation of rat liver nuclei with undegraded rapidly labeled RNA was established.