Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System
Nucleic acids isolated from a variety of sample types provide the starting material for a broad range of downstream applications. Quantification of isolated nucleic acid is necessary prior to introduction into these applications. The Epoch Spectrophotometer System provides a mean of obtaining accura...
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Brescia, P. Banks, P. Voloshin, А. 2017-04-20T13:16:17Z 2017-04-20T13:16:17Z 2012 Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System / P. Brescia, P. Banks, А. Voloshin // Наука та інновації. — 2012. — Т. 8, № 2. — С. 43-47. — Бібліогр.: 2 назв. — англ. 1815-2066 DOI: doi.org/10.15407/scin8.02.043 https://nasplib.isofts.kiev.ua/handle/123456789/116089 Nucleic acids isolated from a variety of sample types provide the starting material for a broad range of downstream applications. Quantification of isolated nucleic acid is necessary prior to introduction into these applications. The Epoch Spectrophotometer System provides a mean of obtaining accurate measurement over a wide range of sample concentrations and volumes, including micro-volume quantification. Comparison to other micro-volume dedicated devices shows equivalent performance. Нуклеїнові кислоти, що виділяються з різноманітних типів зразків, є вихідним материалом для численних подальших досліджень. Для їх виконання необхідна кількісна оцінка ізольованих нуклеїнових кислот. Спектрофотометр Epoch забезпечує надійні вимірювання у широкому діапазоні концентрацій і об’ємів, у тому числі квантифікацію у мікрооб’ємі, та демонструє відмінні характеристики порівняно до інших пристроїв. Нуклеиновые кислоты, выделяемые из разнообразных типов образцов, являются исходным материалом для многочисленных дальнейших исследований. Для их выполнения необходима количественная оценка изолированных нуклеиновых кислот. Спектрофотометр Epoch обеспечивает надежные измерения в широком диапазоне концентраций и объемов, включая квантификацию в микрообъеме, и демонстрирует отличные характеристики в сравнении с другими устройствами. en Видавничий дім "Академперіодика" НАН України Наука та інновації Світ інновацій Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System Мультиоб’ємний аналіз нуклеїнових кислот з використанням спектрофотометричної системи EPOCH™ Мультиобъемный анализ нуклеиновых кислот с использованием спектрофотометрической системы EPOCH™ Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System |
| spellingShingle |
Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System Brescia, P. Banks, P. Voloshin, А. Світ інновацій |
| title_short |
Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System |
| title_full |
Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System |
| title_fullStr |
Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System |
| title_full_unstemmed |
Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System |
| title_sort |
multi-volume analysis of nucleic acids using the epoch™ spectrophotometer system |
| author |
Brescia, P. Banks, P. Voloshin, А. |
| author_facet |
Brescia, P. Banks, P. Voloshin, А. |
| topic |
Світ інновацій |
| topic_facet |
Світ інновацій |
| publishDate |
2012 |
| language |
English |
| container_title |
Наука та інновації |
| publisher |
Видавничий дім "Академперіодика" НАН України |
| format |
Article |
| title_alt |
Мультиоб’ємний аналіз нуклеїнових кислот з використанням спектрофотометричної системи EPOCH™ Мультиобъемный анализ нуклеиновых кислот с использованием спектрофотометрической системы EPOCH™ |
| description |
Nucleic acids isolated from a variety of sample types provide the starting material for a broad range of downstream applications. Quantification of isolated nucleic acid is necessary prior to introduction into these applications. The Epoch Spectrophotometer System provides a mean of obtaining accurate measurement over a wide range of sample concentrations and volumes, including micro-volume quantification. Comparison to other micro-volume dedicated devices shows equivalent performance.
Нуклеїнові кислоти, що виділяються з різноманітних
типів зразків, є вихідним материалом для численних подальших досліджень. Для їх виконання необхідна кількісна оцінка ізольованих нуклеїнових кислот. Спектрофотометр Epoch забезпечує надійні вимірювання у широкому діапазоні концентрацій і об’ємів, у тому числі
квантифікацію у мікрооб’ємі, та демонструє відмінні характеристики порівняно до інших пристроїв.
Нуклеиновые кислоты, выделяемые из разнообразных
типов образцов, являются исходным материалом для
многочисленных дальнейших исследований. Для их выполнения необходима количественная оценка изолированных нуклеиновых кислот. Спектрофотометр Epoch
обеспечивает надежные измерения в широком диапазоне концентраций и объемов, включая квантификацию в
микрообъеме, и демонстрирует отличные характеристики в сравнении с другими устройствами.
|
| issn |
1815-2066 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/116089 |
| citation_txt |
Multi-Volume Analysis of Nucleic Acids Using the EPOCH™ Spectrophotometer System / P. Brescia, P. Banks, А. Voloshin // Наука та інновації. — 2012. — Т. 8, № 2. — С. 43-47. — Бібліогр.: 2 назв. — англ. |
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2025-11-25T15:41:24Z |
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| fulltext |
43
Наука та інновації. 2012. Т. 8. № 2. С. 43—47.
P. Brescia1, P. Banks1, А. Voloshin 2
1 BioTek Instruments, Inc., P.O.Box 998, Highland Park, Winooski, Vermont, USA
2 BioTek in Ukraine, Kiev
MULTI-VOLUME ANALYSIS OF NUCLEIC ACIDS
USING THE EPOCH ™ SPECTROPHOTOMETER SYSTEM
Nucleic acids isolated from a variety of sample types provide the starting material for a broad range of downstream
applications. Quantification of isolated nucleic acid is necessary prior to introduction into these applications. The Epoch
Spectrophotometer System provides a mean of obtaining accurate measurement over a wide range of sample concentrations
and volumes, including micro-volume quantification. Comparison to other micro-volume dedicated devices shows equivalent
performance.
K e y w o r d s: spectrophotometry, nucleic acids, Epoch, Take3, multi-volume analysis.
© P. BRESCIA, P. BANKS, А. VOLOSHIN, 2012
INTRODUCTION
The isolation of DNA and RNA from a wide ran-
ge of samples provides the starting material for a
diverse array of applications. Common samples are:
1. tissues of various origins, including human
biopsies, animal models, formalin-fixed, paraffin-
embedded sections;
2. body fluids including blood and buccal
swabs;
3. eukaryotic and bacterial cells.
The isolated nucleic acid can then be used for
down stream applications such as those illustrated
at the end of the analytical workflows in Figure 1.
Essentially, DNA-based applications involve
sequencing, genotyping, determining gene copy
number and cloning.
For RNA, the goal is gene expression analysis.
This can either be done directly from RNA using
reverse transcriptase in addition to polymerase in
a one step quantitative RT-PCR experiment, or a
cDNA library can be made from the isolated
mRNA. The advantage of the latter is stability of
DNA relative to RNA. This cDNA can then be
either used for quantitative PCR for single-plex
or low multiplexing of expressed genes, using
DNA microarrays for extremely high multiplex-
ing, or even whole transcriptome analysis by deep
sequencing methods.
Quantification of both DNA and RNA early in
the workflows depicted in Figure 1 is necessary
to ensure that relative amounts of nucleic acid
template (i.e. the portion of the isolated DNA or
RNA that is to be amplified and analyzed) and
other reagents, such as enzymes and free nucle-
otides used for amplification (i.e. in PCR or RT-
PCR), are suitable for efficient reactions.
QUANTIFICATION OF BIOMOLECULES
BY UV SPECTROSCOPY
Standard methods of nucleic acid quantificati-
on postisolation include spectrophotometric ab-
sorption and fluorescence when nucleic acids are
chelated with a binding dye. The most common
method remains absorption spectroscopy due to
the peak absorption of DNA and RNA at 260 nm1.
Briefly, a solution of the nucleic acid to be tested
is placed inside a vessel and loaded into a spectro-
44 ISSN 1815-2066. Science and Innovation. T. 8, № 2, 2012
Світ інновацій
photometer and subject to incident light at a pre-
determined wavelength and intensity (Io). The
absorption of UV light is measured as it passes
through the sample by measuring the intensity
(I) at a photo-detector.
The amount of light absorbed is directly related
to the concentration of the sample, pathlength and
extinction coefficient of the biomolecule of in te rest.
The Beer- Lambert Law relates the light ab sor ption
and concentration of the absorbing molecule via the
following equation where A = log Io / I = εlc. ε re-
fers to the extinction coefficient of the analyte
(εdsDNA = 50 ng-cm μL–1; εRNA = 40 ng-cm/ μL), l is
the pathlength (cm) and c is the concentration of
the analyte (ng/μL), as illustrated in Figure 2.
CUVETTE AND MICROPLATE-BASED
INSTRUMENTATION
Cuvette-based spectrophotometers are the gold
standard instrument for performing spectropho-
tometric determinations of nucleic acids. Accu-
racy and precision in the quantification of native
nucleic acids at 260 nm using standard 1 cm path-
length cuvettes is unmatched by other instru-
ments and methods. Yet the cuvette-based spec-
trophotometer does not lend itself to replicate
measurements of many samples. After each analy-
sis, the cuvette must be emptied, washed and re-
loaded with new sample. This is quickly onerous
if numerous samples need to be quantified.
Fig. 1. Genomic workflows for downstream applications for DNA and RNA
Fig. 2. Quantification of biomolecules by UV spectroscopy
is dependent on the absorbance of light as it passes through
the sample. The relationship is described by Beer-Lambert
Law A = εlc
45ISSN 1815-2066. Наука та інновації. T. 8, № 2, 2012
Світ інновацій
To remedy this situation, laboratories have adop-
ted protocols that utilize a 96- or 384-well micro-
plate based format, and suitable microplate inst ru-
mentation compatible with UV absorption spec-
trophotometry. Traditionally, cuvette based mea-
su re ments are performed in a reader that uses
ho rizontal photometry positioned 90 degrees to
the cuvette resulting in a fixed pathlength. Micro-
plate based instrumentation use vertical photom-
etry where the resulting pathlength is due to the
height of the sample volume in a microplate well
through which the sample is read. The result is a
variable pathlength dependent on sample volume
and microplate well dimensions (Figure 3). Path-
lengths can be corrected for in microplate wells
by taking advantage of a small yet significant
peak in absorbance for water at 977 nm. Using
this phenomenon, the pathlength of any aqueous
solution in a microplate well can be determined
by comparing the absorbance of that solution at a
known pathlength (e.g., 1 cm) to the absorbance
in the microplate well. The ratio of the microplate
well absorbance determination to the 1 cm deter-
mination is equal to the pathlength in centime-
ters. The experimental wavelength absorbance
divided by the pathlength results in absorbance
corrected to 1 cm2.
MICRO-VOLUME INSTRUMENTATION
Micro-volume cuvettes are available which al-
low analysis of samples in the microliter range with
fixed pathlengths a fraction of that available with
standard cuvettes. The main advantage of these
micro-volume cuvettes is that nucleic acid samples
do not have to be diluted to be accurately quanti-
fied as the optical density of the measurement is
reduced by the same factor as the pathlength re-
duction. These also have the advantage of being
used with conventional cuvette spectrophotome-
ters. Yet the microvolume cuvettes have the same
disadvantage as standard cuvettes when it comes
to multiple samples and are often difficult to clean.
A popular device for performing micro-volume
quantification of nucleic acids is Thermo Scien-
tific’s NanoDrop. There are several models, but
the most commonly seen in laboratories is the
single channel model. NanoDrop uses a unique
sample retention technology to create a consist-
ent, very short pathlength, which allows for anal-
ysis without need for dilution. A number of other
similar single channel low volume devices are also
available on the market.
EPOCH™ SPECTROPHOTOMETER SYSTEM
FOR MULTI-VOLUME ANALYSIS
The Epoch™ Spectrophotometer System is Bi-
oTek’s solution for researchers who require low
or micro-volume nucleic acid quantification ca-
pability. This sytem includes a Monochromator-
based UV-V.s Microplate Reader and Take3™
Multi-Volume Plate. BioTek’s Take3 Plate pro-
vides a means to perform microvolume quantifi-
cation on multiple samples, as well as 1 cm meas-
urements with a standard cuvette or the BioCell,
a BioTek designed cuvette.
The Take3 plate allows quantification of up to
sixteen 2 μL samples with a nominal pathlength
of 0,5 mm for microvolume analysis.
The shorter pathlength allows for direct meas-
urement of highly concentrated samples typically
yielded by many low volume preparative kits
such as Qiagen’s AllPrep DNA/RNA Mini kits.
Fig. 3. Fixed pathlength using a vertically orientated cu-
vette (l = 1 cm) and variable pathlength obtained when us-
ing microplates. Pathlength is dependent on sample volume
and microplate well dimensions
46 ISSN 1815-2066. Science and Innovation. T. 8, № 2, 2012
Світ інновацій
Additionally, however, a broad range of con-
centrations and volumes can be measured using
the Take3 plate with its ability to accommodate
either cuvettes or BioCells on the plate in addi-
tion to the microspot locations (Figure 4). The
Epoch Microplate Spectrophotometer is used to
make the measurements.
Furthermore, the Epoch Microplate Spectro-
photometer can measure absorbance of samples
in microplates with densities ranging from 6- to
384-wells for a wide range of applications, includ-
ing ELISAs (Figure 5).
METHODS
To test the applicability of the Epoch Spectro-
photometer System to provide accurate quanti-
tation of a variety of sample types, volumes and
concentrations, total DNA and RNA from Chi-
nese Hamster Ovary (CHO-M1) cells from cul-
ture were isolated. Purified samples were then
quantified using the Epoch Spectrophotometer
Fig. 4. The Take3 plate provides various options to measure
samples: a) 16 microspot locations for 2 μl sample loading for
micro-volume analysis. b) the plate can accommodate two
BioCells and one standard cuvette (each 1 cm pathlength)
Fig. 5. Multi-Volume analysis with Epoch™ Spectrophoto-
me ter System
Fig. 6. dsDNA quantification from multi-volume capability of
Epoch Spectrophotometer System and comparison to Nano-
Drop. Percentages illustrated on the columns of the bar chart
reflect % difference of the measurement relative to a 1 cm path-
length measurement made with BioCell in the Take3 plate
Fig. 7. RNA quantification from multi-volume capability of
Epoch Spectrophotometer System and comparison to Nano-
Drop. Percentages illustrated on the columns of the bar chart
reflect % difference of the measurement relative to a 1 cm path-
length measurement made with BioCell in the Take3 plate
a
b
47ISSN 1815-2066. Наука та інновації. T. 8, № 2, 2012
Світ інновацій
System using either undiluted samples in micros-
pot locations on the Take3 plate or after dilution
for concentrated samples in a BioCell and 96-well
microplates. Additional measurements were per-
formed on the NanoDrop 2000c (Thermo Scien-
tific) for comparison.
RESULTS
The dsDNA derived from ~3 X 106 CHO-M1
cells was determined to be at a concentration of ~
440 ng/μL based on all measurements with dif-
ferent methods/instrumentation (see Figure 8).
The sample was eluted from the AllPrep column
in a total volume of ~300 μL elution buffer for a
total yield of approximately 129 μg dsDNA with
an A260/280 ratio of ~1,9 indicating a highly pu-
rified sample preparation. Quantification was ve-
ry consistent across all volumes tested, where ac-
curacies fell within 2 % of the BioCell measure-
ment (Figure 6).
The RNA yield was approximately 1800 ng/μL
in a total volume of 150 μL for a total of approxi-
mately 270 μg RNA with an A260/280 ratio of
~2,0, again indicative of good quality sample pre-
pa ration (Figure 10). Quantification was again
fo und to be consistent across all volumes tested,
where accuracies fell within ~± 2,5% with the
largest deviations derived from measurements
taken in the microplate format (Figure 7).
CONCLUSION
Quantification of nucleic acids after isolation
from biological samples is a standard procedure for
many applications. The Epoch Microplate Spec-
trophotometer, used with the Take3 plate, makes a
system that can provide accurate quantification
from micro-volume, microplate and 1 cm path-
length cuvettes and BioCell. Micro-volume quan-
tification using the microdots of the Take3 plate
obviates the need to dilute the isolated nucleic
acid samples. Up to 16 samples can be quantified
at the same time with the Take3 plate. Compari-
sons to 1 cm pathlength measurements demon-
strate an equivalence of analytical performance.
REFERENCES
1. Sambrook and Russell. Molecular Cloning: A Laboratory
Manual (3rd Ed.). Cold Spring Harbor Laboratory Press.
2001.
2. Held. «Nucleic Acid & Protein in the Microplate Format»
CHIMIA 2001, vol. 55, issue 1—2, p. 40—42.
П. Бреска, П. Бэнкс, А.Н. Волошин
МУЛЬТИОБЪЕМНЫЙ АНАЛИЗ
НУКЛЕИНОВЫХ КИСЛОТ
С ИСПОЛЬЗОВАНИЕМ
СПЕКТРОФОТОМЕТРИЧЕСКОЙ
СИСТЕМЫ EPOCH™
Нуклеиновые кислоты, выделяемые из разнообразных
типов образцов, являются исходным материалом для
многочисленных дальнейших исследований. Для их вы-
полнения необходима количественная оценка изолиро-
ванных нуклеиновых кислот. Спектрофотометр Epoch
обеспечивает надежные измерения в широком диапазо-
не концентраций и объемов, включая квантификацию в
микрообъеме, и демонстрирует отличные характеристи-
ки в сравнении с другими устройствами.
Ключевые слова: спектрофотометрия, нуклеиновые
кислоты, Epoch, Take3, мультиобъёмный анализ.
П. Бреска, П. Бенкс, А.М. Волошин
МУЛЬТИОБ’ЄМНИЙ АНАЛІЗ
НУКЛЕЇНОВИХ КИСЛОТ
З ВИКОРИСТАННЯМ
СПЕКТРОФОТОМЕТРИЧНОЇ
СИСТЕМИ EPOCH™
Нуклеїнові кислоти, що виділяються з різноманітних
типів зразків, є вихідним материалом для численних по-
дальших досліджень. Для їх виконання необхідна кіль-
кісна оцінка ізольованих нуклеїнових кислот. Спектро-
фотометр Epoch забезпечує надійні вимірювання у ши-
рокому діапазоні концентрацій і об’ємів, у тому числі
квантифікацію у мікрооб’ємі, та демонструє відмінні ха-
рактеристики порівняно до інших пристроїв.
Ключові слова: спектрофотометрія, нуклеїнові кис-
лоти, Epoch, Take3, мультиоб’ємний аналіз.
Стаття надійшла до редакції 04.01.12
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