Variations in the physiological state of cells and tissues can be identified using gene expression analysis, which is a well-established process. Previous research has shown that obtaining high-quality RNA from tissue samples is an essential step in obtaining accurate and reliable gene expression data using real-time quantitative PCR (qPCR), hybridisation microarrays, and next-generation sequencing (NGS).
It has long been thought that recovering adequate amounts of high-quality RNA from small (15 mg) and/or complex eukaryotic tissue samples was difficult. Several companies have recently started selling RNA Extraction Kit to recover high-quality RNA from these types of samples. Few studies, therefore, have explicitly compared the efficiency of commercially available kits in isolating RNA from small amounts of DNA. However, few direct comparisons of the performance of commercially available kits in isolating RNA from small tissue samples have been made.
Although the technologies used by commercially available kits to isolate total RNA from tissue samples vary, they all follow the same general procedure:
- lysis of cells and inhibition of ribonuclease (RNase) activity
- DNA elimination
- Complete RNA isolation (of which 1-5 per cent is mRNA).
To lyse cells and inhibit RNase activity, most kits require that tissues be homogenised in a buffer containing detergents and an RNase denaturing agent. The most common method for removing genomic DNA (gDNA) is to use the DNase I enzyme; however, some kits use non-enzymatic methods such as liquid-liquid (e.g., gDNA eliminator solution) extractions and solid-phase extractions.
Numerous methods for RNA isolation are available, with liquid-liquid extraction (e.g., phenol-chloroform, TRIzol, etc.) and solid-phase extraction (e.g., silica-based membrane or paramagnetic bead technologies) being the most widely used by commercially available kits.
While many RNA isolation kits entail manual operation, due to the growing demand for high-throughput assays, kits developed for use with liquid-handling robots are becoming more widely available.
Before downstream gene expression analysis, regardless of the RNA isolation technique used, the quantity of recovered RNA must be determined. RNA can be quantified using spectrophotometry (UV) or fluorometry (RNA specific dye). The amount of RNA available for downstream analysis is determined by the gene expression quantification method used. The amount of total RNA needed for first-strand cDNA synthesis, for example, is in the picogram (pg) to nanogram (ng) range, while the amount required for microarray analysis is frequently in the microgram (ng) range.
Selection Of RNA Extraction Kit
When choosing an RNA extraction kit, consider the kit’s ability to recover the necessary amount of total RNA and the kit’s ability to recover RNA that meets particular quality requirements. The following total RNA samples should be used in gene expression analysis:
- Free of proteins and contaminants that can inhibit downstream molecular analyses
- Intact and free of nucleases (which can contribute to subsequent degradation)
- Free of gDNA contamination.
Determine ratios of sample absorbance at 260 and 280 nm to determine the degree of sample contamination by proteins and other contaminants (A260:A280). Complete RNA samples with values of 1.8 are considered “pure” and suitable for downstream molecular analyses (nucleic acids absorb at 260 nm, while proteins and other contaminants of significance soak at 280 nm), and samples with values of 1.8 are considered “pure” and acceptable for downstream molecular analyses.
A so-called “RNA integrity number (RIN)” or “RNA Quality Indicator (RQI)” has recently become the gold standard for RNA quality evaluation. These metrics indicate the degree of RNA fragmentation (with higher values indicating more intact RNA) and can be obtained using lab-on-chip gel electrophoresis systems based on Caliper Life Sciences’ LapChip® microfluidics technology.
Agilent Technologies (Bioanalyzer2100) and BioRad (Caliper Life Sciences, Inc.) market it (Experion). Most downstream molecular applications consider RNA with RIN or RQI values of 7-8 to be an ideal template.
The minimum appropriate RIN value, on the other hand, is determined by the type of study to be performed. RNA with RIN values of 5-5.5, for example, is a suitable template for qPCR reactions when the amplicon duration does not exceed 200 bp in several studies.
The typical method for determining gDNA contamination is to perform qPCR reactions on samples that are either “no-RT” or “RT(-)” . Since reverse transcription is not performed on RT(-) samples, they can only contain RNA, which cannot be amplified in qPCR reactions. As a result, a fluorescent signal in RT(-) samples suggests gDNA contamination (Jeffries et al., 2014).
Jeffries, M. S., Kiss, A. J., Smith, A. W., & Oris, J. T. (2014). A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. BMC Biotechnology, 14(94). https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-014-00948