Isolation of high-quality RNA for high throughput applications from secondary metabolite-rich Crocus sativus L.

Abstract

Objective Isolating high-quality RNA is a basic requirement while performing high throughput sequencing, microarray, and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides, and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction including CTAB, TriZol, and SDS-based methods, which invariably yield less and poor quality RNA and hence it necessitated the isolation of high-quality RNA suitable for high throughput applications. Results In the present study we made certain adjustments to the available protocols including modifications in the extraction buffer itself and the procedure employed. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) value greater than 7.5. The quality of the RNA was further assessed by qPCR-based amplification of mRNA and mature miRNAs such as Cs-MIR166c and Cs-MIR396a.

Dissection of paracrine/autocrine interplay in lung tumor microenvironment mimicking cancer cell-monocyte co-culture models reveals proteins that promote inflammation and metastasis

“…Total RNA was isolated from A549 and THP-1 cells as reported in [ 12 ]. Real-time PCR was performed using gene specific primers and SYBR Green (Kapa Biosystems, SA).The reaction mixture was run in lightCycler 480-II (Roche, Germany) and the Gene expression was quantified using 2 −ΔΔCT method [ 13 ].…”

Evaluation and Standardization of RNA Extractions with Quality for RNA-Seq for Balamuthia mandrillaris

“…Only protocols Q3 (Figure 1A), T1 (Figure 1B), T2 (Figure 1C), T3 (Figure 1D) showed good integrity based on the intensity and ratio of the 28S and 18S rRNA bands. RNA is considered of high quality when the 28S subunit has a 2:1 ratio over the 18S subunit in gel electrophoresis [21][22][23], so T1, Q3, T2 and T3 comply with this characteristic. The RNA integrity in electrophoresis gel is evidenced by the presence of 2 kb and 4 kb bands, which indicates that the RNA is not degraded; this is a critical quality control step for experiments such as transcriptome sequencing [24].…”