RNA isolation

The ABCA1 gene encodes for a member of subfamily A of the ATP-binding cassette transporters that plays an important role in cellular export of cholesterol and phospholipids; therefore, quantification of the ABCA1 mRNA is critical in many studies related to its expression and regulation by metabolic factors, nutritional status, and new antiatherogenic drug candidates. We developed a rapid, sensitive, specific, and reproducible real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of ABCA1 transcripts in total RNA isolated from cultured human cells and tissues. To quantify ABCA1 mRNA, we generated a calibration curve from serial dilutions of in vitro-transcribed RNA corresponding to an amplified ABCA1 cDNA 205-bp fragment (homologous calibrator). Two pairs of fluorescent hybridization probes were used to detect the ABCA1 and porphobilinogen deaminase (PBGD) mRNAs; the latter served as an internal control. PCR was performed as real-time amplification of A...

A substantial problem when isolating total RNA from human whole blood is the susceptibility to degradation and inconsistent yield of RNA especially when isolation is performed from clinical samples. Here, we report an improved method for isolating high integrity of total RNA from human peripheral blood using guanidium thiocyanate-phenol-chloroform. We found that this cost effective method was able to consistently produced high yield and high integrity of total RNA with average RNA concentration > 2000 ng/mL of blood and RNA integrity number (RIN) > 8.0, as well as negligible genomic DNA contamination from human whole blood that was comparable to other commercial blood total RNA isolation kits. In addition, the present method is also suitable for blood sample that required up to 24 hr transportation time prior total RNA extraction, and able to obtain consistent result in real-time polymerase chain reaction (qPCR) downstream application.

We have investigated the expression of Mr 53,000 protein (p53) in total RNA isolated from human peripheral blood mononuclear cells stimulated by phytohemagglutinin, in serum-stimulated human diploid fibroblasts, and in normal and tumor cells of human epithelial colon tissue. We have found that the expression of p53 messenger RNA is growth regulated in human cells following kinetics similar to that previously shown in mouse 3T3 cells, and is increased in the large majority of colon adenocarcinomas in comparison to adjacent normal mucosa and adenoma. This increased expression of p53 is accompanied by a nearly proportional increase in the expression of histone H3. As the expression of histone H3 is restricted to the S phase of the cell cycle and therefore measures the growth fraction of a given population, we suggest that the increased expression of p53 observed in the large majority of colon tumors simply reflects the increased number of cycling cells frequently found in a neoplastic ...

A series of simple and phenol-free silica-based protocols for isolating and cleaning RNA or DNA fragments from different sources were developed. Cytoplasmic RNA isolated from hybridoma cells by this method was used in RT-PCR. DNA fragments obtained using this protocol were suitable for further subcloning, gene transformation and DNA sequencing. © Rapid Science Ltd. 1998

Due to the presence of a diverse array of metabolites, no standard method of RNA isolation is available for plants. We noted that polysaccharide and polyphenol contents of cardamom tissues critically hinder the RNA extraction procedure. Hence, we attempted several methods for obtaining intact mRNA and small RNA from various cardamom tissues. It was found that protocols involving a combination of commercial kits and conventional CTAB (cetyl trimethylammonium bromide) methods yielded RNA with good purity, higher yield, and good integrity. The total RNA isolated through this approach was found to be amenable for transcriptome and small RNA analysis through next-generation sequencing platforms. Molecular techniques such as next-generation sequencing (NGS) 1 have been established as a highly sensitive and efficient method for small RNA and transcriptome analysis. However, the poor-quality RNA samples interfere with the sequencing procedure. The RNA extraction strategy varies in different species based on the levels of secondary metabolites and polysaccharides [1]. There are previous reports on extracting high-purity RNA from plants [2–6], but these methods did not generate intact RNA from tissues of car-damom (Elettaria cardamomum Maton), the plant of our interest. Hence, this study aimed to find out the most ideal protocol for isolation of high-quality RNA from different tissues for downstream applications such as small RNA sequencing and transcriptome analysis using NGS platforms. The plant tissues used for RNA isolation were collected from the Jawaharlal Nehru Tropical Botanic Garden & Research Institute (JNTBGRI) cardamom germplasm conservatory (accession no. CT9). Fresh leaves, stems, flowers, flower buds, and young fruits were collected, washed well with distilled water, and covered with sterilized aluminum foil to avoid RNase contamination and were immediately placed in liquid nitrogen. The tissues remained as such until further processing. Solutions for RNA isolations were treated with 0.01% diethylpy-rocarbonate (DEPC, Sigma) and then incubated overnight at room temperature. The treated solutions were autoclaved for 20 min to degrade the traces of DEPC. All of the reagents used in the protocols were prepared using the DEPC-treated water to inactivate RNases. Materials such as mortar, pestle, and spatula were also treated with DEPC, incubated overnight, wrapped in aluminum foil, and baked at 180°C for 5 h. The workbench for RNA isolation, pipettes, all plasticwares, and centrifuge were wiped twice with RNaseZap (Ambion) to remove RNase present on the surface. Frequent swabbing of gloves with RNaseZap solution avoids RNase contamination. Electrophoresis gel tank, gel comb, and gel tray were soaked in buffer (0.1 M NaOH and 100 mM ethylenediaminete-traacetic acid) for 2 h and rinsed well with DEPC-treated water. The tissues were subjected to nine RNA isolation methods: modified CTAB (cetyl trimethylammonium bromide) method (hereafter referred to as CTAB method) [7], TRIzol method [8], TRIzol with sodium sulfite method [9], Qiagen's RNeasy Plant and miRNeasy Mini Kits, PureLink RNA Mini Kit (Ambion), and the combined methods of PureLink RNA Mini Kit and CTAB, miRNeasy Mini Kit and CTAB (Dongyan Zhao, Michigan State University, personal communication), and RNeasy Plant Mini Kit and CTAB. The RNA isolation method based on CTAB implemented in this study was a modification of the previously described protocol [7] with a change in the amount of initial sample (100 mg of leaves and stems or 250 mg of flowers, flower buds, and young fruits), incubation time (30 min for leaves and stems or 40 min

Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 °C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.

We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5'-rapid amplification of cDNA ends, deep sequencing of 3' cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5'-RNA adapter that includes an MmeI recognition site is ligated to 5'-monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5' equally-sized fragments are gel-selected, ligated to a 3' double-stranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high...

Citation: Elekwachi CO, Wang Z, Wu X, Rabee A and Forster RJ (2017) Total rRNA-Seq Analysis Gives Insight into Bacterial, Fungal, Protozoal and Archaeal Communities in the Rumen Using an Optimized RNA Isolation Method. Advances in high throughput, next generation sequencing technologies have allowed an in-depth examination of biological environments and phenomena, and are particularly useful for culture-independent microbial community studies. Recently the use of RNA for metatranscriptomic studies has been used to elucidate the role of active microbes in the environment. Extraction of RNA of appropriate quality is critical in these experiments and TRIzol reagent is often used for maintaining stability of RNA molecules during extraction. However, for studies using rumen content there is no consensus on (1) the amount of rumen digesta to use or (2) the amount of TRIzol reagent to be used in RNA extraction procedures. This study evaluated the effect of using various quantities of ground rumen digesta and of TRIzol reagent on the yield and quality of extracted RNA. It also investigated the possibility of using lower masses of solid-phase rumen digesta and lower amounts of TRIzol reagent than is used currently, for extraction of RNA for metatranscriptomic studies. We found that high quality RNA could be isolated from 2 g of ground rumen digesta sample, whilst using 0.6 g of ground matter for RNA extraction and using 3 mL (a 5:1 TRIzol : extraction mass ratio) of TRIzol reagent. This represents a significant savings in the cost of RNA isolation. These lower masses and volumes were then applied in the RNA-Seq analysis of solid-phase rumen samples obtained from 6 Angus X Hereford beef heifers which had been fed a high forage diet (comprised of barley straw in a forage-to-concentrate ratio of 70:30) for 102 days. A bioinformatics analysis pipeline was developed in-house that generated relative abundance values of archaea, protozoa, fungi and bacteria in the rumen and also allowed the extraction of individual rRNA variable regions that could be analyzed in downstream molecular ecology programs. The average relative abundances of rRNA transcripts of archaea, bacteria, protozoa and fungi in our samples were 1.4 ± 0.06, Frontiers in Microbiology | www.frontiersin.org 1