An optimized step-by-step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high-quality intact total RNA

An optimized step-by-step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high-quality intact total RNA

The degeneration of intervertebral disk is the most significant, and least understood, causing chronic back pain, which affects almost one of seven individuals at a certain time. Each intervertebral disc has three components; Central Nucleus Pulposus (NP), concentric layer Annulus fibrosus (AF), and a pair of end ends (EP) that connects the disk to the vertebral body.

Understanding the molecular and cellular basis of intervertebral disc growth, health and aging will result in significant information to develop a therapeutic approach. Preparation of efficient and efficient and efficient pure pure cells is very important for meaningful and strict downstream analysis at cellular, molecular, and biochemical levels. Cross-sample contamination can affect the interpretation of results. In addition to changing gene expression, slow or delayed isolation procedures will also cause cell degradation and biomolecules that create bias in the results of the study.

The mouse model system is used extensively to understand the biology of intervertebral printing. Here we explain two protocols: (a) for efficient insulation from NP, AF, and EP cells from lumbar mouse intervertebral disks. We validate the purity of NP and AF cells using SHH CRE / +; R26 MT / MG / + MICE Dual-fluorescent reporter where all NP cells are GPF + VE, and with a sensitive approach to QPCR analysis using Taqman probes for SSH, and brachyury as a specific marker of the NP, TenomMulin as a special marker AF, and Osteocalcin as a marker Bone specific. (B) for high-quality intact RNA isolation with Rin 9.3 to 10 of the disk cell. These methods will be useful for strict analysis of NP and AF cells, and enhance our understanding of the biology of intervertebral print.

Method of two steps for high quality RNA isolation from the corn seeds that are stored rich in starch

The modified SDS-Trizol method is optimized for total RNA isolation from corn seeds stored periodically one month for 4 months. Use of SDS extraction buffers before the use of trizol reduces co-precipitation problems associated with high carbohydrate content in seeds. Recording average RNA results from seeds across the storage interval is 978.6 ± 65.46 ng / μL. The average spectrophotometric value (260/280) of the isolated RNA varies from 1.974 ± 0.033 to 1.998 ± 0.022. Efforts to isolate RNA from green leaves using the Trizol method also ensure quality and quantity comparable to isolated RNA.

The results of the RNA from fresh leaves were recorded at 1008.2 ± 77,088 ng / μl which were slightly higher than the average RNA results from the seeds throughout the month. Observed an average 260/280 isolated RNA value was 1.984 ± 0.030. Dnase care increasingly increases the ratio of 260/280 in both seeds (2,003 ± 0.006) and leaves (2.012 ± 0.037). High quality and quantity along with isolated RNA integrity are confirmed through the downstream analysis after RNA extraction such as the first cdna strand synthesis and normal PCR. RNA extraction of seeds stored using SDS-based trizol methods that are modified and from fresh leaves using the Trizol method opens up the new possibility of understanding the role of the main genes involving development measures, especially in the development of development, especially in the development of development.

An optimized step-by-step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high-quality intact total RNA

Nuclei isolation from fresh frozen brain tumors for single nucleus RNA-SEQ and Atac-SEQ

Adult diffuse glioma shows intern and intra-tumor heterogeneity. Until now, most of the profiling efforts of large-scale molecules have focused on the bulk approach that causes molecular classification of brain tumors. Over the past five years, a single cell sequencing approach has highlighted some important glioma features. The majority of these studies have used fresh brain tumor specimens to isolate a single cell using a flow cytometry or antibody-based separation method. Moving forward, the use of fresh frozen tissue samples from Biobank will provide greater flexibility for single cell applications.

Furthermore, as a single cell advance advance, the next challenge is to produce multi-omics data from one single cell or the same sample preparation to uncover the complexity of a better tumor. Therefore, a simple and flexible protocol that allows data making for various methods such as Single Nucleus RNA sequencing (SNRNA-SEQ) and a single nucleus test for accessible chromatin transposes with high-throughput sequencing (SNATAC-SEQ) will be important for the field. , The latest advances in the field of single cells coupled with accessible microfluidic instruments such as the 10X genomic platform have facilitated single cell applications in the research laboratory.

To study the heterogeneity of brain tumors, we developed an enhanced protocol for single nuclei isolation from fresh frozen gliomas. This protocol combines the existing single cell protocol and combines homogenization steps followed by filtration and mediated gradient centrifugation. The sample produced is a pure single nuclei suspension that can be used to produce a single nucleus gene expression and chromatin accessibility data from the same nuclei preparation.

Total RNA isolation uses monophasic lysis reagents

Typical mammal cells contain ~ 10-5 μg of RNA, 80% -85% of them are RNA Ribosomes (RRNA; especially species of 28s, 18s, 5.8, and 5s). Most of the remaining 15% of -20% consist of a variety of low molecular weight species (eg, RNAS [tras] and small nuclear RNAS).

This abundant RNA has a specified size and sequence. Conversely, RNA Messenger (MRNA), which forms between 1% and 5% of total cellular RNA, heterogeneous in both sizes – from several bases to many long kilobases and sequences. In this introduction, the development and use of monophasic lysis reagents for total Isolation of RNA from eukaryotic cells is discussed.

Kenneth Hill