High-density Hapten Labeling and HRP Conjugation of Oligonucleotides for Use as In Situ Hybridization Probes to Detect mRNA Targets in Cells and Tissues

Abstract

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.

Molecular Assembly of Superquenchers in Signaling Molecular Interactions

“…This self-quenched probe does not fluoresce until hybridized to its target because of spatial separation of the quencher and the fluorophore. In our preparation, SQs were synthesized by assembling different numbers and different types of quencher molecules using dendrimeric linkers. − The SQs were then used to label MBs. Three fluorescein (FAM)-labeled MBs with the same sequence (quencher-CC TAG CTC TAA ATC ACT ATG GTC GCG CTA GG-FAM) were prepared.…”

In Situ Hybridization AT-Tailing with Catalyzed Signal Amplification for Sensitive and Specific in Situ Detection of Human Immunodeficiency Virus-1 mRNA in Formalin-Fixed and Paraffin-Embedded Tissues

“…Although the sensitivity of ISH-AT-CSA has not reached a level at which it can be used to detect integrated viral DNA (provirus), this issue might be resolved using a cocktail of several HybrAT probes or by developing a new HybrAT probe similar to the branched oligonucleotide probe reported previously. 5 In conclusion, the above data suggest that ISH-AT-CSA can be used as an alternative to the RISH-CSA. The ISH-AT-CSA method is significantly simplified and should be applicable broadly in the fields of cell biology and pathology.…”

A novel method for depositing erythroid cells onto glass slides for fetal cell analysis

“…In previous work in our laboratory, cytospin preparations were dried and fixed in 4% formaldehyde ϩ 5% acetic acid in PBS, with a final pH of 3.5 (21). These preparations gave strong fluorescence signals in subsequent in situ hybridizations (19). Using a cocktail of epsilon-globin and gamma-globin probes ( Table 2) to identify cells of the primitive and definitive lineages in fetal cord blood on similar cytospin preparations, we saw an average of 30% *Flaskette slides were treated with HCl, washed with PBS, coated with poly-L-lysine, anti-glycophorin A (␣-Gpa), or nothing, and washed with PBS.…”

“…Slides were prepared and fixed using modifications of procedures described by Luehrsen et al (19). Cells were deposited onto Shandon-coated slides using a Shandon cytospin (Shandon Lipshaw, Pittsburgh, PA), dried, and fixed in PBS/FA/AA, consisting of PBS with 4% formaldehyde (EM Science, Gibbstown, NJ) and 5% acetic acid (Sigma), pH 3.5, for 20 min at room temperature.…”