Labeling type and detection method
As previously mentioned, ISH probes are labeled with nucleotides modified either with a fluorophore or an antigen. This difference is linked to the detection method.
Fluorophore-bearing probes allow for a
direct fluorescence detection in fluorescence
in situ hybridization (FISH). FISH is a highly sensitive technique, allowing the use of different fluorophores to visualize multiple target sites on the same specimen. FISH is useful for chromosome mapping and enumeration, as well as for the detection of structural rearrangements such as translocations, inversions, insertions and deletions. However, FISH can be limiting when information on tissue morphology is needed, thus a chromogenic detection may then be preferred.
CISH (Chromogenic
in situ hybridization) is typically associated with an
indirect detection and the use of biotin- or digoxigenin labeled probes.
The vitamin
Biotin has been the first replacement to radiolabeled nucleotides, as it was possible to exploit its high affinity binding to
avidin or
streptavidin, in turn conjugated to a reporter enzyme [alkaline phosphatase (AP) or horseradish peroxidase (HRP)] for color development. Biotin can also be recognized by a specific antibody, with a downstream detection system pretty much identical to IHC/IF. However, biotin probe labeling can cause
staining issues, in particular in tissues endogenously expressing this vitamin in high quantity, such as liver, kidney, skeletal and cardiac muscle, etc.
In response to this problem,
digoxigenin-conjugated probes represent a valid alternative. Digoxigenin is a steroid antigen derived from plants of the genus
Digitalis (3). Since it is found exclusively in these plants, it circumvents the possible background issue associated with biotin. The detection occurs via the specific binding with an
anti-digoxigenin antibody. In addition, digoxigenin and biotin labeling can be combined in multiplexing, for instance, two different
in situ probes can be used simultaneously, or if chromogenic ISH is combined with IHC.
Advancements in RNA ISH for single molecule visualization
Early diagnosis and
personalized treatments are two of the major challenges that modern medicine faces. For this reason, the quantitative and qualitative analysis of DNA, RNA, and protein biomarkers is becoming more and more prominent in clinical practice, providing crucial information in diagnosis, prognosis, and therapy guidance
(4). While DNA-based ISH and IHC are commonly applied in this context, the use of RNA-based ISH is still restricted to highly expressed genes (e.g. EBER1/2)
(4), the main reason of that being the sensitivity of the technique, especially when it comes to the detection of single copy targets. For example, by using “classic” RNA ISH, the detection of HPV in cervicovaginal smears can be easily missed, as infections are often caused by a single integration of the viral genome in a small number of cells within a specimen
(5). Therefore, RT-PCR is generally considered the gold standard for gene expression analysis in clinical diagnostic (as well as in any research field), since it allows the detection of very limited amount of the sequence of interest. Nonetheless, differently from ISH, RT-PCR requires the homogenization of the sample for DNA/RNA extraction, and consequently it cannot provide any spatial information concerning gene expression.
In order to overcome these limitations, continuous improvements on the ISH techniques have been introduced over the years, in an attempt to increase its sensitivity. The most common approach consists in enhancing the detection through the use of
branched DNA (bDNA) (4,6), whose most popular application utilizes
pairs of Z-probes (or double Z-probes). As illustrated in Fig. 2, a Z-probe is composed of three elements: a sequence complementary to the target RNA (necessary for the hybridization); a
spacer (linker), and the
Z-probe tail. The latter pairs with the
pre-amplifier sequence, which in turn binds to multiple
amplifiers. Finally, several lab
eled probes (chromogenic or fluorescent) attach to their specific sites on the amplifier molecules (7). The specificity of the system is assured by the fact that detection can occur only if both Z-probes are simultaneously annealed to the target, while the signal amplification process allows for an extremely elevated sensitivity.
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Figure 2. Schematic representation of the pairs of Z-probes amplification technology.
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Despite the numerous evident advantages of this form of bDNA technologies, some of its aspects can still constitute a constraint. For instance, given the multiple steps required for the amplification, the protocol is considerably long. The hybridization conditions can also be harsh, thereby altering tissue or affecting the possibility to visualize other markers. Finally, its overall elevated cost makes it less accessible to smaller laboratories.
AMPIVIEW™ RNA probes
Enzo Life Sciences, considered a pioneer in labeling and detection since the development of the first non-radioactive human papillomavirus (HPV) probes for ISH in 1986, is today proud to give a further contribution in the field with the introduction of the
AMPIVIEW™ RNA probes, based on the patented
LoopRNA technology. As represented in figure 3, in a LoopRNA ISH probe (LRP), the regions pairing with the target sequence are interspersed with multiple
RNA spacer segments, which are not complementary to the target. The spacers are designed so that most of their bases can be labeled with either biotin or digoxigenin. When such a probe is hybridized to its target sequence, the labeled spacer segments loop out and can then be recognized by the reporter system
(5). The presence of
multiple molecules of biotin or digoxigenin, easily
accessible for the reporters thanks to the loops, allow for a
strong amplification of the signal, thus assuring an
excellent sensitivity. Since the hybridization does not need to undergo multiple amplification steps, the protocol is much
faster and easier compared to the bDNA system described above. In addition, the AMPIVIEW™ probes do not require any specific equipment, and can be used in manual or automated procedures.
Concerning the detection, pretty much as for a standard ISH, it can be direct or indirect. When the probe is labeled with biotin, a streptavidin conjugated with a reporter enzyme can be directly used (
direct or one-step protocol). Enzo’s
SAVIEW® PLUS AP or HRP Reagent is a ready-to-use streptavidin-based nanopolymer reagent, ensuring consistent and reproducible results. In the
indirect (or two-step) protocol, anti-biotin or anti-digoxigenin linker antibodies can be combined with ready-to-use nanopolymer
POLYVIEW® PLUS AP or HRP detection reagents, producing high intensity color development, with sharp and crisp staining. Stunning results can then be achieved when combining SAVIEW® PLUS or POLYVIEW® PLUS reagents with Enzo’s unrivaled selection of
HIGHDEF® chromogens (9). For all these reasons, AMPIVIEW™ RNA probes represent a
cost-effective solution and easily adaptable into any laboratory.
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Figure 3. Simplified diagram of the AMPIVIEW™ RNA Probes design and how signal is amplified.
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Faithful to our historical background, the first AMPIVIEW™ RNA probes generated were to target HPV. In a recent collaboration between Enzo and two Italian universities, Dr. La Rocca
et al. demonstrated that ISH probes based on the LoopRNA technology allow the detection of HPV DNA and RNA even down to a single copy of genome-integrated HPV. These results are compared with another commercially available double-Z probe, demonstrating the equivalent or superior performance of the AMPIVIEW™ RNA probes
(5). The article also highlights another relevant feature of LoopRNAs, which is the possibility to synthetize probes corresponding to both
sense (complimentary to the viral genomic DNA) and
antisense sequence (complimentary to genomic DNA and RNA transcript) in order to distinguish between genomic DNA and RNA being actively transcribed by the virus.
It is also important to mention here that AMPIVIEW™ RNA probes can be designed for practically any gene, in any genome. For instance, during the COVID-19 pandemic, Dr. Nuovo from Ohio University used an AMPIVIEW™ probe to detect SARS-CoV-2 RNA expression in different tissues from COVID-19 patients, comparing the presence of viral genome with the presence of the four characteristic viral protein (envelope, spike, membrane, and nucleocapsid)
(8). Dr. Nuovo has also been a valuable guest speaker in webinar series to illustrate these results. You can re-watch its brilliant talk
HERE!
Do you have questions regarding our AMPIVIEW™ RNA probes, powered by Enzo’s LoopRNA™ technologies? If you are looking for a probe for a specific target and would like to test the AMPIVIEW solution, do not hesitate to reach out to our
Technical Support Team. We will be happy to assist!
