Application Note

Effects of low A260/A230 ratios in RNA preparations on

downstream applications

The efficiency of applications such as real-time RT-PCR and RNA-seq depends strongly on the
purity of the RNA sample used. To assess RNA purity, the absorbance of RNA at 260 nm and the
absorbance of potential contaminants at 280 nm or 230 nm was determined by spectroscopic
measurement (e.g., QIAxpert® or NanoDrop®). An A260/A280 ratio of 1.8–2.1 at pH 7.5 is widely
accepted as indicative of highly pure RNA. Pure RNA should also yield an A260/A230 ratio of
around 2 or slightly higher; however, there is no consensus on the acceptable lower limit of this
ratio. Also, it has not been fully established whichA contaminants contribute to a low A260/A230
ratio. Possible candidates include salt, carbohydrates,
A /Apeptides and aromatic compounds such as
260 230

phenol (1, 2). In our experience, increased absorbance

2.5 at 230 nm in RNA samples is almost
always due to contamination by guanidine thiocyanate,
2.0 a salt which absorbs very strongly at
220–230 nm and can be present at very high concentrations
1.5 in the lysis buffer or extraction
GITC
reagent (e.g., TRIzol®) used in most RNA purification
1.0
procedures. Our experiments showed
GuHCIthat

the A260/A230 ratio of an RNA sample is strongly reduced when guanidine thiocyanate is present
0.5
even at submillimolar concentrations (Figure 1A). However, we also found that concentrations
0.0
of guanidine thiocyanate of up to 100 mM in an RNA
0.01sample 0.1
do not compromise
1 the reliability
10 100
Guanidine
of real-time RT-PCR, even when using PCR chemistries that are sensitive salt (mM) (Figure 1B).
to inhibitors
Similar observations have been reported by other researchers (3).
A B
A260/A230 CT value
2.5 40
GITC 1 µl
2.0 35 GITC 2 µl
GuHCI 1 µl
1.5 30 GuHCI 2 µl
GITC
1.0 GuHCI 25

0.5 20

0.0 15
0.01 0.1 1 10 100 0 1 10 100 1000
Guanidine salt (mM) Guanidine salt (mM)

Figure 1. Effect of guanidine salt concentration on the A260/A230 ratio and real-time RT-PCR. (A) Ratio of A260 to A230 for RNA
samples (50 ng/μl) containing 0.03–100 mM guanidine hydrochloride (GuHCl) or 0.01–30 mM guanidine thiocyanate
B
(GITC). (B) CT values obtained from real-time one-step RT-PCR using a TaqMan® Gene Expression Assay for beta-actin and a
master
CT value from Supplier AII. The reaction volume was 25 μl, and the template was either 1 μl or 2 μl of a 50 ng/μl RNA
mix
sample containing 0.3–1000 mM guanidine salt.
40
GITC 1 µl
35 GITC 2 µl
GuHCI 1 µl
30 GuHCI 2 µl

25

20

Sample
15 to Insight
0 1 10 100 1000
Guanidine salt (mM)
 Impact of RNA concentration and contaminants on the A260/A230 ratio
When establishing a suitable lower limit for the A260/A230 ratio, it is important to remember that this
ratio (and other absorbance ratios in general) also depends on RNA concentration. Trace amounts
of contaminants will have virtually no effect on the ratio if the RNA is at a high concentration, but
will have a major impact on the ratio if the RNA concentration is low. However, the most important
factor is the amount of contaminant that is transferred to the downstream reaction (e.g., cDNA
synthesis), rather than the absorbance ratio.

Example with low mass input samples

A low A260/A230 ratio can also be influenced by a low starting sample amount in the RNA extraction
procedure, leading to a low concentrated RNA sample after extraction. Despite recommendations
to use a certain amount of starting material, sometimes the amount is limited due to the source of
material or availability (e.g., tissue, biopsies, etc.). Smaller sample amounts can therefore result in
lower A260/A230 ratios in comparison to recommended starting amounts. The RNA samples below
(from 5 or 0.5 mg brain extracted using the miRNeasy Micro Kit either manually or a QIAcube®
for automated purification procedure) had a normal A260/A280 ratio around 1.8–2.1, but reduced
A260/A230 ratios especially for the 0.5 mg samples (Table 1). The A260/A230 ratios for the low mass
input samples were within 0.8–1.5.

OD 280 nm OD 260 nm A260/A280 A260/A230 ng/μl Vol. Yield Mw Std. Dev.

10.653 5.159 2.06 1.81 426.1 13.5 5.752

QIAcube 5.731 0.022
10.573 5.139 2.06 1.81 422.9 13.5 5.709
5 mg
Manual 10.089 4.889 2.06 1.75 403.6 11.5 4.641
4.702 0.060
10.354 5.05 2.05 1.95 414.1 11.5 4.762

1.036 0.512 2.02 1.51 41.46 14.5 0.601

QIAcube 0.615 0.014
1.122 0.576 1.95 0.85 44.87 14.0 0.628
0.5 mg
1.25 0.625 2 1.29 50.02 11.5 0.575
Manual 0.647 0.072
1.563 0.88 1.78 0.89 62.54 11.5 0.719

Table 1. Effect of standard and low mass sample input on A260/A230 ratio calculated from absorbance measurements made
on a Nanodrop® spectrophotometer. RNA from different amounts of brain tissue were extracted using the miRNeasy® Mini
Kit either manually or on the QIAcube.

A low A260/A230 ratio does not influence downstream applications

Although the A260/A230 ratio is low, no influence or inhibition is observed in downstream applications,
such as RT-PCR (Figure 2A). In our experiments, the real-time PCR run for the samples with a low
A260/A230 ratio resulted in typical amplification (Figure 2B) and melting curves (Figure 2C). The
ΔCT for both sample input amounts was approximately 3.3, independent of the extraction procedure.
Although this analysis was performed using real-time RT-PCR, similar conclusions apply to microarray
analysis and other applications that rely on cDNA synthesis as the first step — the step that is the
most influenced by contaminants.

2 Effects of low A260/A230 ratios in RNA preparations on downstream applications 01/2018

A

CT sample 1 CT sample 2 Mw Std. Dev

QIAcube 14.569 14.318 14.444 0.126

5 mg
Manual 14.409 14.323 14.366 0.043

QIAcube 17.769 18.068 17.919 0.150

0.5 mg
Manual 17.697 17.612 17.655 0.043

B C
Amplification Plot Melt Curve Plot
Derivative
ΔRn Reporter (–Rn)

0.20
0.1
0.24

0.19
0.01
0.14

0.001 0.09

0.04
0.0001
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 65.0 70.0 75.0 80.0 85.0 90.0 95.0

Cycle Tn: 76.4

Figure 2. Detection of mir16 in a SYBR Green RT-PCR Assay. (A) CT values obtained from real-time one-step RT-PCR using a
®

miScript® Gene Expression Assay for mir16. The same sample template volumes were used for all reactions. (B) Amplification
plots and (C) melting curves showed comparable curves and a single melting peak, indicating homogeneous amplification.

QIAxpert ® and Spectral Content Profiling

The QIAxpert is an innovative µ-volume UV/Vis spectrophotometer that overcomes limitations of
classic spectrophotometry and purity assessment using absorbance ratios.

Using reference spectra of known contaminants, a state of the art software algorithm on the
QIAxpert instrument performs a deconvolution of measured UV/Vis spectra according to the Beer
Lambert law for mixtures, stating that the absorption spectrum of a mixture is a linear combination
of the spectra of its individual constituents. This feature, known as Spectral Content Profiling
(SCP), allows dye-free and easy differentiation between DNA, RNA and other UV/Vis absorbing
contaminants in complex biological samples.

The QIAxpert enables yield and purity assessment in a single measurement while offering accurate
dye-free DNA, RNA fractions quantification irrespective of the buffering conditions and assessment
of the amount of contributing quantities of co-purified substances including sample turbidity,
contaminating nucleic acids, phenol and other contaminants present in the sample. Our experiments
showed that it is possible to get reliable RNA quantification despite increasing guanidine thiocyanate
contamination and decreasing A260/A230 ratios in 50 ng/µl RNA (Figure 3). However, guanidine
thiocyanate in RNA in applied concentrations for QIAxpert measurements had no influence on
CT values obtained with real-time one-step RT-PCR (Figure 1B).

Effects of low A260/A230 ratios in RNA preparations on downstream applications 01/2018  3

 Sample RNA Nucleic acids Impurities Background
Position name (ng/μl) (ng/μl) (A260) (A260) Residue (%) A260 A260/A280 A260/A230

A1 RNA 49.9 49.9 0.00 1.10 1.10 1.24 1.80 2.34

RNA + 0.3
B1 50.9 50.9 0.00 0.74 0.74 1.28 2.06 0.74
mM GITC
RNA + 0.6
C1 50.7 50.7 0.02 0.96 0.96 1.28 2.03 0.54
mM GITC

A1 (RNA) B1 (RNA + 0.3 mM GITC) C1 (RNA + 0.6 mM GITC)

Absorbance (10 mm) Absorbance (10 mm) Absorbance (10 mm)

1.4 1.4 1.4

1.2 1.2 1.2

1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0.0 0.0 0.0

230 260 290 320 350 230 260 290 320 350 230 260 290 320 350
Wavelength (nm) Wavelength (nm) Wavelength (nm)

Figure 3. QIAxpert measurements with RNA. RNA concentration and purity were measured on QIAxpert with and without spiked-in guanidine thiocyanate
contamination. (Top) Results table and (bottom) spectral content profiling spectra, showing total measured spectrum (black); RNA spectrum (blue); impurities
spectrum, including guanidine thiocyanate (orange); residues (yellow); and background (gray). The RNA concentration was the same regardless of
increasing guanidine thiocyanate contamination.

Note: Some sources have reported that the absorbance maximum for guanidine thiocyanate is
around 260 nm (4), which is similar to that for RNA (Figure 4A). This observation appears to be due
to a saturation effect on the Nanodrop spectrophotometer at high concentrations of the salt, about
10–1000 times higher compared to what may be observed in RNA isolated by GTC-based
methods (Figure 4A). This would incorrectly lead to an optimal A260/A230 ratio and an overestimation
of RNA concentration at high GITC contamination on a Nanodrop. At moderate or low salt
concentrations, the absorbance maximum is around 220–230 nm, leading to low A260/A230 values.
On other spectrophotometers, such as the SpectraMax® or the QIAxpert, the right shoulder of the
GITC absorption spectrum shifts to the right at higher concentrations, due to saturation, while the
absorbance maximum remains below 230 nm wavelength (Figure 4B–C). In conclusion, while
QIAxpert can estimate RNA concentrations precisely despite moderate contamination with GITC
(Figure 3), at very high GITC concentrations, QIAxpert and SpectraMax would overestimate
RNA concentrations with low A260/A230 values. The Nanodrop would also overestimate RNA
concentration and incorrectly show good A260/A230 values.

4 Effects of low A260/A230 ratios in RNA preparations on downstream applications 01/2018

A Figure 4. Effect of high guanidine thiocyanate concentrations
Nanodrop
on absorbance measurements made on (A) Nanodrop
Absorbance (10 mm) spectrophotometer, (B) Spectramax and (C) QIAxpert.
15.32 The wavelength for peak absorbance appears to increase
at high salt concentrations on a Nanodrop, while SpectraMax
14.00 0.1 mM GITC and QIAxpert showed a shift of the right shoulder due to
12.00 1 mM GITC saturation.
10 mM GITC
10.00
100 mM GITC
8.00
1000 mM GITC
6.00

4.00

2.00

0.00
- 1.53
220 230 240 250 260 270 280 290 300 310 320 330 340 350
Wavelength (nm)

B Spectramax
Absorbance (10 mm)

2.5

0.1 mM GITC
2.0 1 mM GITC
10 mM GITC
1.5 100 mM GITC
1000 mM GITC
1.0

0.5

0.0
220 250 280 310 340
Wavelength (nm)

C QIAxpert
Absorbance (10 mm)

40

35 0.1 mM GITC
1 mM GITC
30
10 mM GITC
25
100 mM GITC
20 1000 mM GITC
15

10

0
230 260 290 320 350
Wavelength (nm)

Effects of low A260/A230 ratios in RNA preparations on downstream applications 01/2018  5

 References
1. Teare JM, Islam R, Flanagan R, Gallagher S, Davies MG, Grabau C. (1997) Measurement of nucleic acid concentrations
using the DyNA Quant and the GeneQuant. Biotechniques. 6, 1170. www.bcm.edu/mcfweb/?PMID=3100
2. G
 allagher SR. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. (2011) Curr Protoc Mol
Biol. 10.1002/0471140864.psa04ks52.
3. C
 icinnati, V.R., Shen, Q., Sotiropoulos, G.C., Radtke, A., Gerken, G., and Beckebaum, S. (2008) Validation of putative
reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR.
BMC Cancer. 8, 350.
4. T hermo Scientific (2010). T042‐Technical Bulletin NanoDrop Spectrophotometers, 260/280 and 260/230 Ratios
[online].

Ordering Information
Product Contents Cat. no.
miRNeasy Micro Kit (50) For 50 total RNA preps: 50 RNeasy MinElute Spin Columns, ®
217084
Collection Tubes (1.5 ml and 2 ml), QIAzol® Lysis Reagent,
RNase-Free Reagents and Buffers
QIAcube (110 V) Robotic workstation for automated purification of DNA, RNA, or 9001292
proteins using QIAGEN spin-column kits: includes 1-year warranty
on parts and labor
QIAxpert QIAxpert instrument with 1 year warranty coverage including 9002340
parts, labor and shipping
miRNA16miScript Primer Assay (100) 10x miScript Primer Assay (contains one miRNA-specific primer) Varies
miScript II RT Kit (12) For 12 cDNA synthesis reactions: miScript Reverse Transcriptase 218160
Mix, 10x miScript Nucleics Mix, 5x miScript HiSpec Buffer,
5x miScript HiFlex Buffer, RNase-Free Water
miScript SYBR Green PCR Kit (200) For 200 reactions: QuantiTect ® SYBR Green PCR Master Mix, 218073
miScript Universal Primer

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN
Technical Services or your local distributor.

Visit www.qiagen.com/geneglobe.

Trademarks: QIAGEN®, Sample to Insight ®, QIAcube®, QIAxpert ®, QIAzol®, MinElute®, miScript®, QuantiTect®, RNeasy® (QIAGEN Group); TaqMan® (Roche Group); Nanodrop® (Thermo Fisher Scientific); SpectraMax®
(Molecular Devices, LLC); SYBR® (Life Technologies Corporation); TRIzol® (Molecular Research Center, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to
be considered unprotected by law.
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