Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence
Provided is a method for direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence to measure RNA in a sample. Also provided is a method of diagnosing a disease in a subject, wherein the disease is caused by increased or decreased expression of a causative gene.
1. A method of measuring an amount of target ribonucleic acid (RNA), comprising
a. incubating an RNA sample with one or more fluorescently labeled RNA probes complementary to one or more respective target RNA sequences in the RNA sample under conditions whereby the one or more probes can hybridize to the one or more respective target RNAs to form one or more RNA:fluorescently labeled probe hybrids;
b. passing the RNA sample comprising the one or more RNA:fluorescently labeled probe hybrids through a capillary electrophoresis system;
c. detecting and recording fluorescence as one or more peaks as the RNA sample comprising the one or more RNA:fluorescently labeled probe hybrids passes through a detection window, wherein each of the one or more RNA:fluorescently labeled probe hybrids generates its own peak; and
d. determining an area under each peak for the respective RNA:fluorescently labeled probe hybrid whereby the area under each peak corresponds to the amount of respective target RNA in the RNA sample, wherein no target RNA is reverse transcribed during steps (a)-(d).
2. The method of claim 1 , wherein RNA is isolated from a cell, a tissue, or an organ of a subject.
3. The method of claim 1 , wherein the one or more fluorescently labeled RNA probes are made by incorporating a fluorophore into the one or more RNA probes during a synthesis reaction.
4. The method of claim 2 , wherein the amount of target RNA measures gene expression in the cell, the tissue, or the organ.
5. The method of claim 1 , wherein the one or more peaks of step (c) correspond to one or more transcripts of a gene.
6. The method of claim 1 , wherein the one or more peaks of step (c) correspond to distinct species of RNA.
7. The method of claim 1 , wherein the fluorescently labeled probe comprises a fluorophore selected from the group consisting of Alexa Fluor 488-5-UTP, BODIPY TR-14-UTP, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6-tetramethyl rhodamine), the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, 6-carboxyfluorescein (6-FAM), 2′,4′,1,4,-tetrachlorofluorescein (TET), 2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamine (JOE), 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1,4-dichloro-6-carboxyfluorescein (NED), and 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC).
8. The method of claim 1 , wherein the one or more fluorescently labeled probes hybridize to RNA under stringent conditions.
9. The method of claim 1 , wherein the capillary electrophoresis system is a microfluidic device.
10. The method of claim 1 , wherein the capillary electrophoresis system is equipped with laser detection, comprising one or more lasers.
11. The method of claim 10 , wherein a first laser is an argon laser having a wavelength from about 470 nm to about 510 nm.
12. The method of claim 11 , wherein the wavelength is 488 nm.
13. The method of claim 10 , wherein a second laser has a wavelength from about 610 nm to about 650 nm.
14. The method of claim 13 , wherein the wavelength is 635 nm.
15. The method of claim 1 , wherein the one or more peaks are recorded in an electropherogram.
16. A method of measuring an amount of target ribonucleic acid (RNA), comprising
a. incubating an RNA sample with one or more fluorescently labeled RNA probes complementary to one or more respective target RNA sequences in the RNA sample under conditions whereby the one or more probes can hybridize to the one or more respective target RNAs to form one or more RNA:fluorescently labeled probe hybrids;
b. passing the RNA sample comprising the one or more RNA:fluorescently labeled probe hybrids through a capillary electrophoresis system;
c. detecting and recording fluorescence as one or more peaks as the RNA sample comprising the one or more RNA:fluorescently labeled probe hybrids passes through a detection window, wherein each of the one or more RNA:fluorescently labeled probe hybrids generates its own peak; and
d. determining an area under each peak for the respective RNA:fluorescently labeled probe hybrid whereby the area under each peak corresponds to the amount of respective target RNA in the RNA sample, wherein no target RNA is reverse transcribed during steps (a)-(d), and wherein the amount of RNA is calculated from either the following equation (a):
[
m
RNA
]
=
[
probe
]
×
A
target
A
target
+
A
probe
,
(
a
)
wherein [probe] is the concentration of fluorescently labeled RNA probe used in a hybridization reaction, wherein A target and A probe are the area under the curve for a target RNA:fluorescently labeled probe hybrid peak and an unhybridized probe peak respectively, whereby the concentration of mRNA, [mRNA], in the sample is calculated; or from the following equation (b):
Area
=
Area
max
×
Mass
probe
K
d
+
Mass
probe
(
b
)
wherein K d equals the mass of a fluorescently labeled RNA probe needed to reach half saturation of a target RNA by the fluorescently labeled RNA probe, wherein Mass probe is the mass of a fluorescently labeled RNA probe used in a hybridization reaction, wherein Area max is the maximum area obtained when all of the target RNA is bound by the fluorescently labeled RNA probe, whereby the amount of target RNA present can be calculated from the K d value using the following expression:
mass
RNA
=
2
×
K
d
×
(
molar
mass
target
RNA
molar
mass
probe
)
.