Genomics Core Facility of the University of Leicester Technical note March 2012

Transcription

1 Genomics Core Facility of the University of Leicester Technical note March 2012 Consequences of various contaminants on RNA quantification and quality controls Nicolas Sylvius and Mariaelena Repici Department of Genetics, University of Leicester, UK RNA isolation is performed everyday by molecular biologists for a wide range of applications. No matter what protocol is used and how meticulous the user is, a risk to degrade RNA during the process or to carry over contaminants always exists which may affect the reliability of the subsequent analytical techniques. The parameters that are usually checked for quality control include the concentration, the ratios of absorbance A260/280 and A230/260 on a spectrophotometer and the RNA Integrity Number (RIN) used by the Agilent Bioanalyzer. However, biologists new in the field of RNA are often puzzled when these parameters vary from the expected optimal values. The most common problems are RNA partially degraded and RNA contaminated by protein, genomic DNA, or reagents used during purification. Here we provide examples of Bioanalyzer electropherograms and nanodrop values resulting from various type of contamination or degradation. These can be used for guidance to identify the causes of problems in RNA preps. We envisaged: Contamination by TRIzol Contamination by protein Contamination by ethanol Contamination by genomic DNA Various levels of RNA degradation Method: Aliquot of 10 µl of high quality mouse RNA was contaminated with 5 µl of either RNAse free water (control aliquot), TRIzol, 100% ethanol, BSA (stock solution 1mg/ml), genomic DNA (stock solution 30 ng/µl). Two control aliquots were also degraded 15 minutes at 70 C or 85 C. Quantification was performed using a Nanodrop 8000 and an Agilent 2100 bioanalyzer with the Agilent RNA Nano kit. Contaminantion by TRIzol: This is probably one of the most detrimental contaminants. On a spectrophotometer, concentrations are largely under estimated. The A260/280 ratio is massively reduced and the ratio A230/260 is reduced or returns aberrant values. On the Bioanalyzer electropherogram all the peaks, including the lower marker at 25 nt, are massively reduced, which results in a concentration aberrantly low (Figure 1, 2). The RIN number cannot be determined. The reduced size of the lower marker peaks at 25nt, which should always be around 15 to 25 FU, is a hallmark of TRIzol remaining (Figure 2). Figure 1. Agilent Bioanalyzer electropherogram of a high quality control RNA sample before (Red) and after (Blue) contamination by TRIzol

2 Figure 2. Close up of the TRIzol contamination trace Control RNA Control RNA + TRIzol Bioanalyzer Conc 353 ng/µl 22 ng/ µl RIN 9.2 N/A Nanodrop Conc 464 ng/ µl 58 ng/ µl A260/ A230/ Contamination by protein On the spectrophotometer, both A260/280 and A230/260 ratios are reduced. The concentration is overestimated. The A260/280 is lowered as proteins also absorb at 280 nm wavelength. The A230/260 ratio is lowered as well. On the Bioanalyzer trace, the 18s and 28s peaks are lowered and the concentration is underestimated (Figure 3). The RIN number is not significantly affected.

3 Figure 3. Agilent Bioanalyzer electropherogram of a high quality control RNA sample before (Red) and after (Green) contamination by proteins Control RNA Contaminated RNA Bioanalyzer Conc 353 ng/µl 153 ng / µl RIN Nanodrop Conc 464 ng/µl 575 ng/µl A260/ A230/ Contamination by ethanol Contamination by ethanol has little to no effect on the A260/280 and A230/260 ratios. Concentrations measured either by absorbance or by the Bioanalyzer are slightly over estimated (Figure 4). However, the RIN number is not affected. Figure 4. High quality control RNA sample before (Red) and after contamination by ethanol (Blue)

4 Control RNA Contaminated RNA Bioanalyzer Conc 353 ng/µl 445 ng/µl RIN Nanodrop Conc 464 ng/µl 548 ng/µl A260/ A230/ Contamination by genomic DNA On the Bioanalyzer, the RIN number is reduced compared to a pure RNA sample. However, the consequences on the electropherogram may not be visible if the contamination is limited and a slightly sub-optimal RIN number may easily be taken as a slight degradation (Figure 5). However, when the contamination is more important, for example in case of large amount of DNA in a low concentrated RNA sample, the contamination is more obvious (Figure 6). The 28s peaks is replaced by a bell-shaped curve and the 18s peak is significantly reduced. The RIN number is greatly affected. Little to no effect is observed on the A260/280 and A260/230 ratios. Figure 5. Agilent Bioanalyzer electropherogram of a high quality control RNA sample before (Red) and after (Blue) contamination by genomic DNA

5 Figure 6. Agilent Bioanalyzer electropherogram of a High quality control low concentrated RNA sample before (Red) and after (Blue) contamination by genomic DNA Control RNA Contaminated RNA Low concentrated RNA contaminated by genomic DNA Bioanalyzer Conc 353 ng/µl 372 ng/μl 52 ng/µl RIN Nanodrop Conc 464 ng/µl 471 ng/µl 63 ng/µl A260/ A230/ RNA degradation High concentrated RNA samples are less sensitive to degradation than low concentrated RNA ones (Figure 7, 8) as highlighted by the RIN number which significantly drops in case of a low concentrated sample as opposed to a highly concentrated one. Harsh degradation significantly reduces the 28s peak and results in a bell shaped curve around the 18s peak (Figure 9). We found the concentration indicated by the Bioanalyzer largely overestimated. On a spectrophotometer the A260/280 and A230/260 ratios are not affected at all. Figure 7. Agilent Bioanalyzer electropherogram of a High quality control RNA sample before (Red) and after (Blue) slight degradation

6 Figure 8. Agilent Bioanalyzer electropherogram of a high quality, low concentrated control RNA sample before (Red) and after (Blue) slight degradation (Blue) Figure 9. Agilent Bioanalyzer electropherogram of a high quality control RNA sample before (Red) and after (Blue) significant degradation Control RNA Slight RNA degradation Slight RNA degradation Large RNA degradation

7 (low Concentration RNA) Bioanalyzer Conc 353 ng/µl 401 ng/µl 32 ng/µl 1012 ng/µl RIN Nanodrop Conc 464 ng/µl 489 ng/µl 53 ng/µl 564 ng/µl A260/ A230/