Reverse transcription polymerase chain reaction (RT-PCR)
is one of many variants of polymerase chain reaction (PCR). This technique is
commonly used in molecular biology to detect RNA expression levels. RT-PCR is often
confused with real-time polymerase chain reaction (qPCR) by students and scientists
alike. However, they are separate and distinct techniques. While RT-PCR is used to
qualitatively detect gene expression through creation of complementary DNA (cDNA)
transcripts from RNA, qPCR is used to quantitatively measure the amplification of DNA
using fluorescent probes. qPCR is also referred to as quantitative PCR, quantitative realtime PCR, and real-time quantitative PCR.
Although RT-PCR and the traditional PCR both produce multiple copies of
particular DNA isolates through amplification, the applications of the two techniques are
fundamentally different. The traditional PCR is simply used to exponentially amplify
given DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing
the RNA of interest into its DNA complement through the use of reverse transcriptase.
Subsequently, the newly synthesized cDNA is amplified using traditional PCR.
In addition to qualitatively study gene expression, RT-PCR can be utilized for
quantification of RNA, in both relative and absolute terms, by incorporating qPCR into
the technique. The combined technique, described as quantitative RT-PCR or real-time
RT-PCR (sometimes even quantitative real-time RT-PCR), is often abbreviated as qRTPCR, RT-qPCR, or RRT-PCR. Compared to other RNA quantification methods, such as
northern blot, qRT-PCR is considered to be the most powerful, sensitive, and quantitative
assay for the detection of RNA levels. It is frequently used in the expression analysis of
single or multiple genes, and expression patterns for identifying infections and diseases.
The western blot (sometimes called the protein immunoblot) is a widely accepted
analytical technique used to detect specific proteins in the given sample of tissue
homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D
structure or denatured proteins by the length of the polypeptide. The proteins are then
transferred to a membrane (typically nitrocellulose or PVDF), where they are stained
with antibodies specific to the target protein.[1][2]
There are now many reagent companies that specialize in providing antibodies (both
monoclonal and polyclonal antibodies) against tens of thousands of different proteins.[3]
Commercial antibodies can be expensive, although the unbound antibody can be reused
between experiments. This method is used in the fields of molecular biology,
biochemistry, immunogenetics and other molecular biology disciplines.
Other related techniques include using antibodies to detect proteins in tissues and cells by
immunostaining and enzyme-linked immunosorbent assay (ELISA).
A general blotting procedure[4] starts with extraction of total RNA from a homogenized
tissue sample or from cells. Eukaryotic mRNA can then be isolated through the use of
oligo (dT) cellulose chromatography to isolate only those RNAs with a poly(A) tail.[7][8]
RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the
probes are unable to enter the matrix, the RNA samples, now separated by size, are
transferred to a nylon membrane through a capillary or vacuum blotting system.
Northern Blot
A nylon membrane with a positive charge is the most effective for use in northern
blotting since the negatively charged nucleic acids have a high affinity for them. The
transfer buffer used for the blotting usually contains formamide because it lowers the
annealing temperature of the probe-RNA interaction, thus preventing RNA degradation
by high temperatures.[9] Once the RNA has been transferred to the membrane, it is
immobilized through covalent linkage to the membrane by UV light or heat. After a
probe has been labeled, it is hybridized to the RNA on the membrane. Experimental
conditions that can affect the efficiency and specificity of hybridization include ionic
strength, viscosity, duplex length, mismatched base pairs, and base composition.[10] The
membrane is washed to ensure that the probe has bound specifically and to avoid
background signals from arising. The hybrid signals are then detected by X-ray film and
can be quantified by densitometry. To create controls for comparison in a northern blot,
samples not displaying the gene product of interest can be used after determination by
microarrays or RT-PCR
Southern Blot
1. Restriction endonucleases are used to cut high-molecular-weight DNA strands into
smaller fragments.
2. The DNA fragments are then electrophoresed on an agarose gel to separate them by
size.
3. If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may
be treated with an acid, such as dilute HCl, which depurinates the DNA
fragments, breaking the DNA into smaller pieces, thus allowing more efficient
transfer from the gel to membrane.
4. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution
(typically containing sodium hydroxide) to denature the double-stranded DNA.
The denaturation in an alkaline environment may improve binding of the
negatively charged thymine residues of DNA to a positively charged amino
groups of membrane, separating it into single DNA strands for later hybridization
to the probe (see below), and destroys any residual RNA that may still be present
in the DNA. The choice of alkaline over neutral transfer methods, however, is
often empirical and may result in equivalent results.[citation needed]
5. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or
below, depending on the direction of the transfer) the gel. Pressure is applied
evenly to the gel (either using suction, or by placing a stack of paper towels and a
weight on top of the membrane and gel), to ensure good and even contact between
gel and membrane. If transferring by suction 20X SSC buffer is used to ensure a
seal and prevent drying of the gel. Buffer transfer by capillary action from a
region of high water potential to a region of low water potential (usually filter
paper and paper tissues) is then used to move the DNA from the gel on to the
membrane; ion exchange interactions bind the DNA to the membrane due to the
negative charge of the DNA and positive charge of the membrane.
6. The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours
(standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet
radiation (nylon membrane) to permanently attach the transferred DNA to the
membrane.
7. The membrane is then exposed to a hybridization probe—a single DNA fragment with
a specific sequence whose presence in the target DNA is to be determined. The
probe DNA is labelled so that it can be detected, usually by incorporating
radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In
some cases, the hybridization probe may be made from RNA, rather than DNA.
To ensure the specificity of the binding of the probe to the sample DNA, most
common hybridization methods use salmon or herring sperm DNA for blocking
of the membrane surface and target DNA, deionized formamide, and detergents
such as SDS to reduce non-specific binding of the probe.
After hybridization, excess probe is washed from the membrane (typically using SSC
buffer), and the pattern of hybridization is visualized on X-ray film by autoradiography in
the case of a radioactive or fluorescent probe, or by development of color on the
membrane if a chromogenic detection method is used.
Enzyme-linked immunosorbent assay (ELISA) is a test that uses antibodies and color
change to identify a substance.
ELISA is a popular format of a "wet-lab" type analytic biochemistry assay that uses a
solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually
an antigen, in a liquid sample or wet sample.
The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as
a quality-control check in various industries.
Antigens from the sample are attached to a surface. Then, a further specific antibody is
applied over the surface so it can bind to the antigen. This antibody is linked to an
enzyme, and, in the final step, a substance containing the enzyme's substrate is added.
The subsequent reaction produces a detectable signal, most commonly a color change in
the substrate.
Performing an ELISA involves at least one antibody with specificity for a particular
antigen. The sample with an unknown amount of antigen is immobilized on a solid
support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to
the surface) or specifically (via capture by another antibody specific to the same antigen,
in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is
added, forming a complex with the antigen. The detection antibody can be covalently
linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an
enzyme through bioconjugation. Between each step, the plate is typically washed with a
mild detergent solution to remove any proteins or antibodies that are not specifically
bound. After the final wash step, the plate is developed by adding an enzymatic substrate
to produce a visible signal, which indicates the quantity of antigen in the sample.
Of note, ELISA can perform other forms of ligand binding assays instead of strictly
"immuno" assays, though the name carried the original "immuno" because of the
common use and history of development of this method. The technique essentially
requires any ligating reagent that can be immobilized on the solid phase along with a
detection reagent that will bind specifically and use an enzyme to generate a signal that
can be properly quantified. In between the washes, only the ligand and its specific
binding counterparts remain specifically bound or "immunosorbed" by antigen-antibody
interactions to the solid phase, while the nonspecific or unbound components are washed
away. Unlike other spectrophotometric wet lab assay formats where the same reaction
well (e.g. a cuvette) can be reused after washing, the ELISA plates have the reaction
products immunosorbed on the solid phase which is part of the plate, so are not easily
reusable.