Quality of total RNA samples is upmost important to the success of miRNA array assays. Three quality controls are performed including quantity, purity, and integrity.
The quantity and purity of RNA samples can be evaluated using a benchtop spectrometer. UV absorption at 260 nm is used to measure RNA quantity and absorbtion ratios (260/280 ratio and 260/230 ratio) are used as a measure of sample purity.
Absorption 260/230 ratio ≥ 1.0 and 260/280 ratio ≥ 1.8 are used as indicators of acceptable purity.
- Low 260/280 ratios are often attributed to phenol and/or protein contaminations.
- Low 260/230 ratios are usually attributed to salt (e.g. guanidine isothiocyanate) and/or phenol contaminations. Contaminants may cause serious downstream problem by prohibiting enzymatic labeling reactions.
- “High-salt”, seen as 260/230 ratio less than 1.0, is an often occurring contamination problem that causes a significantly increased absorption at 260 nm and result in overestimate of RNA quantity.
Grossly erroneous quantity measurements will lead to significant RNA aliquot variations, increase experiment-induced variations, and reduce high confident calls in profiling data.
RNA sample integrity can be evaluated using Agilent Bioanalyzer 2100 (For total RNA quality analysis, RNA 6000 Pico LabChip kit should be used by following corresponding Agilent User Manual.)
- A high quality eukaryotic RNA sample should have a band intensity ratio of 28S rRNA to 18S rRNA of two.
- Bioanalyzer or 1-1.5% agarose gel
- 28S rRNA band at 4.5kb – ~2X intensity
- 18S rRNA band at 1.9kb.
Electrophoretic traces from Bioanalyzer contain additional information relating to RNA integrity. Agilent introduced an analysis algorithm, RIN (RNA Integrity Number) to standardize RNA integrity interpretation. RIN ranges from 1 to 10, with 1 being the most degraded profile and 10 being the most intact.
- For Average Cell Line or Tissue sample – total RNA with RIN ≥ 8 is generally considered of acceptable integrity for mRNA as well as miRNA profiling analysis.
- For other sample types such as Blood or Plant – RIN number does not apply
Important Note – These quality measures largely reflect the integrity of large RNAs (not small RNA) which count for ~95% by weight of total RNA and dominate electrophoretic trace signals.
It is reasonable to expect cases when UV Ratios and RINs do not reflect integrity of miRNAs in total RNA samples.
Please call us if you are uncertain about your sample quality.
RNA sample integrity can also be evaluated using one of several standard denaturing gel electrophoresis methods. Bioanalyzer is preferred due to its low sample amount requirement and automated quality assessment.*
- Faint or missing bands at low molecular weight area indicates a failure to recover small (including micro) RNA.
- Excessive smearing on the gel indicates degraded RNA.
* Electrophoresis images courtesy of Norgen Biotek Corp.
More information about LC Sciences miRNA profiling, discovery and analysis services is available at: http://www.lcsciences.com/ag-genomics/applications/transcriptomics/mirna-profiling/