High quality RNA isolation directly from the desired network to get the right information from the profiles of gene in vivo expression in cells embedded in their extracellular matrix (ECM). It is known that the RNA purification of the cartilage tissue is very challenging because of the low cell content (condrocytes) and the solid ECM which is rich in negatively charged proteoglys that can be returned with RNA. Older methodology to purify RNA from cartilage that involves the use of concentrated denaturation solutions containing Isothiocyanate guanidinium followed by ultracentrification in trifluoroacetate cesium.
Such an ultracentrification approach is rarely used now since the emergence of a mini column chromatography kit is more user friendly. For this chapter, we test and compare three methods to isolate RNA from murine-art-prone bones (femoral heads) who are immature and find that the combination of Trizol® reagent and chromatography column spin (total purification kit) is the best approach to produce more quality high. RNA. Here, the average RNA integrity number (RIN), as determined by Bioanalyzer technology, is 7.1.
We then apply this method to try to isolate RNA directly from human articular cartilage harvested from three osteoarthritic knee combined specimens (OA). As expected, the concentration and quality of RNA obtained differently between samples. However, from one specimen, we can isolate around 3 μg of the total RNA (including small non-calcoding RNA) of the 100 mg of OA cartilage with Rin = 7.9. Apart from the patient-to-patient variability that is known to exist between the specimens of cartilage from the OA connection, we have shown that it is possible to obtain high levels of RNA from human articular cartilage on the quality that is suitable for downstream analysis including microarray and RNA -EQ. A detailed explanation of our preferred RNA purification methodology, which can be used to isolate the RNA from human networks, cattle, or cartilago, provided in this chapter.
Method for Isolation of RNA Messenger from biological samples
RNA molecules contain many chemical modifications that can regulate various biological processes. Molecules Messenger RNA (MRNA) is an important component in the dogma center of molecular biology. The discovery of reversible chemical modification in Eukaryotics MRNA presents a new field of research in gene expression regulations mediated by RNA modification. Modifications to MRNA are generally in low abundance. The use of very pure MRNA is very important for confidential identification of new modifications and for accurate quantification of existing MRNA modifications.
In addition, very pure MRNA isolation is the first step in many biological research studies. Therefore, the method to isolate MRNA is very pure important for MRNA-based downstream studies. Various methods to isolate MRNA have been developed in recent decades and new methods continue to appear. This review focuses on the methodology and protocol to isolate the population of MRNA. In addition, we discuss the advantages and limitations of this method. We hope this paper will provide general views on MRNA insulation strategies and facilitate studies involving modifications and functions of MRNA.
Fast and easily accessible method for RNA, DNA, and protein isolation to facilitate SARS-COV-2 detection
Pandemic management Covid-19 requires widespread SARS-COV-2 testing. The main limitation for widespread SARS-COV-2 testing is a global lack of important supply, including the RNA extraction kit. The need for commercial RNA extraction kits places obstacles to tests that detects SARS-COV-2 genetic materials, including PCR-based reference tests. Here we propose an alternative method that we call Pearl (Precipitation Enhanced Analyte Retrieval) which address this limitation.
Pearl uses a lysis solution that interferes with cell membranes and temporary virus envelopes simultaneously provide conditions suitable for RNA, DNA, and alcohol-based protein precipitation. Pearl is a fast, cheap and simple method that uses general laboratory reagents and offers comparable performance with commercial RNA extraction kits. Pearl offers alternative methods to isolate nucleic acid and host proteins and pathogens to streamline DNA and RNA virus detection, including SARS-COV-2.
Fecal RNA isolation (micro)
It becomes clear that RNA is in the lumen intestine and dirt in animals and humans. The protocol described below isolated total RNA including Microrna from animal samples and human subjects. The aim is to isolate total RNA with high purity and quantity for downstream analysis such as RNA, RT-PCR, and micro-array sequencing. The advantage of this optimized protocol in MIRNA isolation is the ability to isolate very pure RNA products with additional washing steps described, an increase in the number of RNA obtained by an enhanced method in sample resilings, and important tips from decontamination.
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One limitation is the inability to process and purify samples greater than more than 200 mg because this sample size will cause difficulties in the formation of clear interfases. As a result, large sample sizes can pollute the aqueous phase that will be extracted as described in the protocol with organic things that affect the quality of RNA isolated in the end. However, RNA isolates from samples up to 200 mg are enough for most downstream analysis.