RNAscope™ ISH Technology Workflows
Similar to solving a jigsaw puzzle, successfully performing the technique of in situ hybridization (ISH) benefits from a systematic, step-wise approach that concentrates on one stage of the process at a time, then moves onto the next.
ACD’s RNAscope ISH Technology kits represent a huge advance in the power and ease of use of ISH, greatly simplifying and streamlining time-tested methods while routinely delivering single-molecule detection levels of sensitivity. While not as simple as doing a four-piece jigsaw puzzle, we have made ISH considerably easier than it was formerly. The first piece is sample preparation.
Proper preparation of samples is the foundation for achieving the high levels of sensitivity and specificity possible with RNAscope ISH technology. The ACD in situ hybridization kits (RNAscope, BaseScope™, miRNAscope™, and DNAscope™) can be used with formalin-fixed, paraffin-embedded (FFPE) samples, fresh-frozen (FF) or fixed frozen tissues, and both adherent and non-adherent cultured cells.
Guidance on best practices in preparing each of the above sample types for assay categories (chromogenic, fluorescent, multiplexed, automated) is available on the ACD website. Product manuals and relevant tech notes containing detailed sample prep advice for individual assays are found in the Product Documents section of Support. The Troubleshooting Guide and FAQ section of Support Solutions also contains a wealth of information on sample prep.
Following appropriate sample preparation, the next step in solving the puzzle is to provide sufficient access for in situ hybridization probes to their targets. This is accomplished by specialized pretreatment of properly prepared samples before applying the RNAscope, BaseScope, miRNAscope, or DNAscope probes. Not only must cell membranes be permeabilized for best results, but the crosslinking of nucleic acids to proteins that occurs during preparation of fixed samples can mask targets and must be relieved. And in the case of ACD’s popular chromogenic ISH assays, it is also necessary to block the activity of endogenous peroxidases.
RNAscope ISH Pretreatment reagents
RNAscope Pretreatment reagents provide the right tools for these important jobs. The proprietary formulations of these reagents have been optimized to provide enhanced access of in situ hybridization probes to nucleic acid targets across a wide variety of tissue sample types including FFPE, fresh‑frozen (FF), and fixed frozen tissue as well as tissue microarray (TMA) and cultured adherent and non-adherent cells.
- RNAscope Target Retrieval Reagent: A buffer system used along with heating of the samples to undo the cross-linking that occurs from tissue fixation, including perfused tissue. This reagent is not required for fresh (unfixed) frozen tissue.
- RNAscope Hydrogen Peroxide Reagent: For blocking endogenous peroxidase activity with use of the chromogenic kits.
- The various protease reagents serve to permeabilize cell membranes as well as to unmask RNA or DNA targets by subjecting the proteins to which they were formerly crosslinked to appropriate levels of proteolysis:
- RNAscope Protease Plus Reagent: A mild protease treatment used with the chromogenic kits for FFPE and fixed frozen tissue.
- RNAscope Protease III Reagent: A moderate protease treatment used with the chromogenic kits for cultured cells and with the fluorescent kits for fixed frozen tissue as well as cultured cells.
- RNAscope Protease IV Reagent: A strong protease treatment used with both the chromogenic and fluorescent kits for fresh-frozen tissue.
- RNAscope kits already include pretreatment reagents appropriate for their most typical use, but certain combinations of kits and tissues will require additional reagents.
- RNAscope 2.5 Universal Pretreatment Reagents Kit: Includes all the above the pretreatment reagents to cover the widest variety of sample types.
As with sample preparation, comprehensive advice on best pretreatment practices for different sample types in particular assay categories are available on the ACD website. Product manuals and relevant tech notes containing detailed pretreatment protocols for individual assays are found in the Product Documents section of Support. The Troubleshooting Guide and FAQs section of Support Solutions is also an excellent source of information on how to achieve optimal pretreatment of valuable samples.
Once properly prepared samples have been appropriately pretreated, the next piece of the puzzle involves the application of chromogenic or fluorescent in situ hybridization probes specific to the target of interest.
RNAscope Probe Hybridization Technology
RNAscope, BaseScope, and DNAscope assays employ a probe hybridization strategy that involves pairs of probes, each of which contains 18 to 25 bases complementary to a section of the target RNA adjacent to that of the other pair member. The overall system is designed such that both probes in each pair must hybridize independently to their respective target sequences for subsequent signal amplification to occur.
High Specificity, Excellent Signal-to-Noise Ratio
This pairing requirement not only has the effect of greatly increasing the signal-to-noise ratio but also ensures high specificity stemming from the extremely low probability of two different probes binding adjacent non-specific sequences. Furthermore, our probe design algorithm is validated to select for optimal hybridization within the recommended assay conditions and minimal cross-hybridization to off-target sequences.
Single RNA Molecule Detection Achieved with Degraded Samples or Less Accessible Targets
Although detection of a single RNA target molecule with an RNAscope assay can be accomplished by the binding of just three pairs of probes for the target, there are 20 separate pairs of probes specific to the target in the assay. This high level of redundancy means that even partially degraded or incompletely unmasked RNA targets may still be detectable at single-molecule resolution. Another feature that contributes to the robustness of RNAscope assays in this respect are the relatively short target regions required, with just 36 to 50 bases of combined target sequence being sufficient for the hybridization of a probe pair.
The final piece of the puzzle is the generation of a highly amplified, readily detected signal indicating the presence (and location) of the target molecule. The unique three-component design of RNAscope, BaseScope, and DNAscope hybridization probes provides the basis for sensitivity capable of detecting even a single RNA or DNA molecule.
Adjacent Z-Probe Pairs Form a Binding Site for the Amplification Apparatus
The 18- to 25-base “lower” portion of each hybridization probe of the pair just discussed that binds specifically and adjacently to the target molecule is connected via a spacer sequence to a 14-base “upper” portion. The spacer sequences cause each probe construct to assume a “Z” shape wherein the two upper portions of a bound pair will also lie adjacently in a line. This upper structure forms a 28-base binding site for a so-called pre-amplifier molecule.
As additional insurance against signals arising from non-specific binding, the pre-amplifier requires both upper portions, i.e., the entire 28-base sequence, to be presented contiguously to bind.
Many Amplifiers Bind Each Pre-Amplifier; Each Amplifier Binds Many Labeled Probes
Once bound to the upper portion of the probe pair, the pre-amplifier provides a kind of scaffolding that bears numerous binding sites for “amplifier” molecules, each of which contains a similar number of binding sites for labeled probes that contain either a chromogenic enzyme or a fluorescent molecule.
The multiplicity of labeled probes arising from the binding of a single molecule is what enables the dramatic levels of signal amplification characteristic of RNAscope assays.
Detection by Microscopy
Detection is accomplished via fluorescent or brightfield microscopic analysis, depending on the type of assay selected. Each punctate dot signal represents a single target molecule. The system can also be automated on partner platforms (Leica Biosystems and Roche Tissue Diagnostics). Single-molecule signals can be quantified on a cell-by-cell basis by manual counting or automated using commercial image processing software packages. We provide technical notes for the open-source image processing programs QuPath, ImageJ, and CellProfiler.
This publication details the core principles behind RNAscope Technology:
Wang F, Flanagan J, Su N, et al. RNAscope: A Novel In Situ RNA Analysis Platform for Formalin-Fixed Paraffin-Embedded Tissues. J of Mol Diagnostics. 2012 Jan; 14(1):22-29.