RNAscope In-situ Hybridization Service

RNAscope® is a proprietary in situ hybridization (ISH) assay based on ACD patented signal amplification and background suppression technology, which advances RNA biomarker analysis in tissues and cells. Unique to this technology, RNAscope® delivers quantitative, sensitive and specific molecular detection of RNA species on a cell-by-cell basis with morphological context in a single assay. This enables researchers to visualize which genes are expressed, localize where they are expressed, and quantify the level of expression. There are multiplexing capabilities that include the potential for combined protein and mRNA localization.  

RNAscope can be used for both bright-field and fluorescence microscopy applications.  The Microscopy Core has the hybridization oven and other essential laboratory equipment necessary for the complete RNAscope procedure allowing the user to visualize their work by using either their own bright-field or fluorescent microscope, or the Core’s Leica TCS SP-8 confocal microscope. This procedure can be done by the users, or the Core will perform the staining for a fee (please inquire).

How It Works:

RNAscope® Technology is a novel in situ hybridization (ISH) assay for detection of target RNA within intact cells. The assay represents a major advance in RNA ISH approaches with its proprietary probe design to amplify target-specific signals but not background noise from non-specific hybridization.

Below are three publications from ACD that will give you a more complete look at the concepts and procedures upon which RNAscope is based.  The core will work with you to determine which expression kit would best fulfill your experimental needs.

Listed below is a technical journal article that may give users valuable information about, and helpful insight into, the RNAscope technology:

RNAscope: A Novel In Situ RNA Analysis Platform for Formalin-Fixed Paraffin-Embedded Tissues.

Wang F,Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y (2012). J of Mol Diagnostics, 14(1):22-29. https://pubmed.ncbi.nlm.nih.gov/22166544/

RNAscope Protocol for single mRNA target using bright field detection:

Day 1 Notes:

  • Refer to tissue preparation (FFPE) before beginning
    • Only put one tissue per slide 
  • Xylene goes in the green clearing agent dish
  • 200 mL of 1X Pretreat 2/Target Retrieval is made with 180 mL of DH2O and 20 mL of 10X Target Retrieval Reagents (DO NOT boil at 100 C for more than 15 minutes)
  • Nonlabeled end aligned towards center

Deparaffinization:

  • Bake slides for 60 min at 60 degrees C in a dry oven
  • Fill the water reservoir in the Oster Steamer to the HI line
  • Place a clear steaming bowl onto the base
  • Place two slide holders in steam bowl, fill one with 200 mL of 1X PRETREAT 2 and the other with 200mL of DH2O
  • Cover the steam holes with tape, except one for thermometer
  • Turn on the steamer and set heating time to 95 min
  • Insert the digital thermometer through the holes of the lid and into container with 1X PRETREAT 2 and allow temp to rise to 98-99 degrees C
  • Place slides in slide rack 
  • Submerge slide rack in xylene 1 and incubate for 5min at RT
  • Submerge slide rack in xylene 2 and incubate for 5 min at RT
  • Submerge slide rack in 100% EtOH and incubate for 3 min at RT
  • Submerge slide rack in 100% EtOH and incubate for 3 min at RT
  • Airdry on dry paper towel for 5 min-overnight at RT
  • Draw hydrophobic barrier (PAP Pen) around the tissue
  • Allow PAP Pen to dry
  • Place slides on clean paper towel under hood 



Pretreatment:

  • Add drops of PRETREAT 1 (hydrogen peroxide) to each section and incubate for 10 min at RT
  • Decant PRETREAT 1 and place in slide rack 
  • Submerge slide rack in DH2O, wash by moving slide rack up and down, repeat with fresh DH2O


Target Retrieval:

(Refer to FFPE: Chapter 3 for Steamer or Appendix B for beaker/hot plate- 700ml one)

  • Add slides to container containing DH2O for 10 seconds to acclimate slides
  • Using Forceps, submerge the slide rack into the PRETREAT 2, then cover steamer with lid
  • Check temp again to ensure it is between 95 degrees C (98 C is best) and 104 degrees C
  • Boil for 15 min for mild and standard conditions or 30 min for extended
  • Immediately submerge hot slide rack in 200 mL DH2O at RT, wash by moving slide rack up and down for 15 sec
  • Transfer to 100% alcohol for 3 min
  • Dry slides in 60 degree incubator or at RT
  • Draw hydrophobic barrier around tissue ( if gone ) and allow to dry for 2 min or overnight at RT

Setup for Day 2:       

  • Preheat oven to 40 degrees C 
  • Place the humidifying paper in the center of the bottom of the tray and add 50 mL of DH2O
  • 30 minutes before beginning and keep tray warm throughout

Pretreatment 3 ( Protease Digestion ):

  • Place slides in Humidity Control Tray
  • Add drops of PRETREAT 3 (Protease Plus) to cover tissue
  • Place Humidity Control Tray in HybEZ oven
  • Incubate for 15 for mild conditions and 30 min for standard/extended at 40 degrees C
  • Vortex slightly and heat TARGET or CONTROL Probes for 10 min at 40 C
  • Decant PRETREAT 3 and place in slide rack (Put Humidity Control Tray back in HybEZ oven)
  • Submerge slide rack in DH2O, wash by moving slide rack up and down, repeat with fresh DH2O

Target Probe Hybridization:

  • Decant DH2O, tap on paper towel if needed
  • Place slides in Humidity Control Tray
  • Add drops of TARGET PROBE or CONTROL PROBE to cover tissue 
  • Put Humidity Control Tray in HybEZ oven and incubate for 2 hours at 40 degrees C

Preparation for Assay

  • 3L of 1x Wash Buffer by adding 2.94L DH2O and 1 bottle (60 mL) of 50x Wash Buffer, warm 50x Wash Buffer up to 40 degrees for 10-20 min before making the 1x Wash Buffer (Can be kept at RT for up to 1mo)
  • 50% Hematoxylin by adding 100mL Gill’s Hematoxylin to 100 mL DH2O (Can be reused up to 1wk) in staining dish
  • Prepare 0.02% ammonia water (bluing reagent) (DO NOT TAKE OUT OF HOOD) by adding 1.43 mL of 1N Ammonia Hydroxide to 250 mL DH2O in beaker, cover with parafilm, mix well and transfer to staining dish
  • Add 200 mL xylene to green dish 
  • Fill two staining dishes with 200 mL of 95% EtOH
  • Prepare 70% EtOH by adding 140mL 95% to 60mL DH2O in staining dish, mix well. 
  • Remove AMP 1-6 from fridge and place at RT
  • Ensure tray and oven at 40 degrees C

Amplification:

  • Decant TARGET OR CONTROL PROBE
  • (DO NOT LET SLIDES DRY OUT DURING THE REMAINING AMPLIFICATION PROCEDURE)
  • (THE FOLLOWING WB WASHES CAN BE DONE IN THE SLIDE RACK, BUT IT IS EASIER TO LEAVE IN HUMIDITY CONTROL TRAY SLIDE HOLDER AND WASH IN THE BIG WASH TRAY) 
  • Amps are stable at RT, so can be left out throughout assay
  • 1 bottle of wash buffer will be used per run
  • Place slides in slide rack and submerge in 200ml 1X WASH BUFFER
  • Incubate for 2 min at RT with frequent agitation
  • Replace with 1X WASH BUFFER and repeat
  • Decant WB
  • Place slides on Humidity Control Tray and put 3-4 drops of AMP 1 on each slide
  • Place Humidity Control Tray in holder and cover with lid for 30 min @ 40 degrees C
  • Place slides in slide rack and submerge in 200ml 1X WASH BUFFER
  • Incubate for 2 min at RT with frequent agitation
  • Replace with 1X WASH BUFFER and repeat
  • Decant WB
  • Place slides on Humidity Control Tray and put 3-4 drops of AMP 2 on each slide
  • Place Humidity Control Tray in holder and cover with lid for 15 min @ 40 degrees C
  • Place slides in slide rack and submerge in 200ml 1X WASH BUFFER
  • Incubate for 2 min at RT with frequent agitation
  • Replace with 1X WASH BUFFER and repeat
  • Decant WB
  • Place slides on Humidity Control Tray and put 3-4 drops of AMP 3 on each slide
  • Place Humidity Control Tray in holder and cover with lid for 30 min @ 40 degrees C
  • Place slides in slide rack and submerge in 200ml 1X WASH BUFFER
  • Incubate for 2 min at RT with frequent agitation
  • Replace with 1X WASH BUFFER and repeat
  • Decant WB
  • Place slides on Humidity Control Tray and put 3-4 drops of AMP 4 on each slide
  • Place Humidity Control Tray in holder and cover with lid for 15 min @ 40 degrees C
  • Place slides in slide rack and submerge in 200ml 1X WASH BUFFER
  • Incubate for 2 min at RT with frequent agitation
  • Replace with 1X WASH BUFFER and repeat
  • Decant WB
  • Place slides on Humidity Control Tray and put 3-4 drops of AMP 5 on each slide
  • Place Humidity Control Tray in holder and cover with lid for 30 min @ RT
  • Place slides in slide rack and submerge in 200ml 1X WASH BUFFER
  • Incubate for 2 min at RT with frequent agitation
  • Replace with 1X WASH BUFFER and repeat
  • Decant WB
  • Place slides on Humidity Control Tray and put 3-4 drops of AMP 6 on each slide
  • Place Humidity Control Tray in holder and cover with lid for 15 min @ RT
  • Place slides in slide rack and submerge in 200ml 1X WASH BUFFER
  • Incubate for 2 min at RT with frequent agitation
  • Replace with 1X WASH BUFFER and repeat
  • (MAKE UP DAB MIXTURE BEFORE DECANTING TO PREVENT DRYING OUT)
  • Decant 1X WASH BUFFER and move to Humidity Control Tray

DAB Colorimetric Reaction

  • Prepare a 1:1 DAB MIXTURE by mixing equal volume of Brown A and Brown B (about 120 microliters per slide, so 2 drops of brown A and Brown B, 4 drops total. MIX WELL) in a microtube
  • Mix thoroughly by inverting tube several times
  • Add 120 microliters of DAB MIXTURE to each tissue
  • Incubate for 10 min @ RT in Humidity control tray because slides must be covered 
  • Decant DAB MIXTURE into a waste container with 20% Bleach (40 mL bleach into 200 mL water) 
  • Immediately put slides into slide rack and submerge into DH2O, wash by moving slide rack up and down, repeat with fresh water

Counterstain (Gills Hematoxylin)

  • Move the slide rack into a dish containing a 50:50 mixture of GILLS HEMATOXYLIN and DH2O
  • Incubate for 2 min @ RT (slides will be purple)
  • Transfer slide rack to fresh DH2O
  • Rinse and replace DH2O until water is clear and tissues remain purple
  • Decant the DH2O and submerge slide rack in BLUING REAGENT or 0.02% Ammonia in DH2O, move up and down until sections turn blue
  • Decant BLUING REAGENT
  • Submerge slide rack in fresh DH2O

Dehydration:

  • Transfer slide rack to 70% EtOH for 2 min (OA)
  • Transfer slide rack to 95% EtOH 1 for 2 min (OA)
  • Transfer slide rack to 95% EtOH 2 for 2 min (OA)
  • Transfer slide rack to 100%EtOh for 2 min (OA)
  • Transfer slide rack to Xylene for 5 min (OA)

Mounting:

  • Put cytoseal over tissue and coverslip with the appropiate size cover glass
  • Allow slides to air dry