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=RNA Scope= [[Daniel Jacobsen|Back to Main]] RNA Scope is a proprietary protocol used by ACD Bio. It can be done using fluorescence (3 channels, maybe 4, +DAPI) or using chromogenic staining (2 colors plus counter stains). ==Manuals== *[[Media:Fresh_Frozen_Sample_Prep_Part_1_UM.pdf|Fresh Frozen Sample Prep User Manual]] *[[Media:RNAscope_Multiplex_UM.pdf|Multiplex Fluorescent Assay User Manual]] *[[Media:320497_RNAscope_2_Part_2_UM-Brown_11132013_a.pdf|BROWN Detection Kit Manual]] *[[Media:Fresh_frozen_Tech_Note_092514.pdf|Fresh Frozen Tissue Prep Quick Guide]] *[[Media:Tech_Note_CulturedCells_Fluor_04092014.pdf|Cultured Cells Manual]] ==Chromogenic Staining Protocol== ===Tissue Prep (~30 minutes)=== #Fixation ##Chill 4 %PFA (Fresh 4% PFA or 10% NBF in 1X PBS) to 4C (ice) ##Remove slides from -80C and immediately immerse in pre-chilled fixative for '''15 minutes''' #Dehydration ##Incubate slides in 50% EtOH for 5 minutes at RT ##Incubate slides in 70% EtOH for 5 minutes at RT ##Incubate slides in 100% EtOH for 5 minutes at RT ##Incubate slides in fresh 100% EtOH for 5 minutes at RT ##Air dry on a paper towel for 5 minutes at RT #Draw a hydrophobic barrier 2-4 times around each section with barrier pen; Let the barrier dry completely (~1 minute) #Turn on oven and set temperature to 40C; wet humidifying paper #Pretreatment ##Place slides on slide rack, add Pretreat 4 to entirely cover each section ##Incubate for 30 minutes at RT ###This time may be considerably shorter for different tissues, recommend 5 minutes for BA8 ###Pretreat 3 is 1/2X Pretreat 4, can provide even gentler pretreat ##Take each slide from rack and tap or flick to remove excess liquid; immediately submerge in 1X PBS ##Wash slides in 1X PBS by moving rack up and down 3-5 times; repeat with fresh 1X PBS ===RNA Scope Assay=== #Place AMP 1-6 reagents at RT; Warm oven to 40C; warm probes for 10 minutes at 40C, swirl gently to mix #Probe Hybridization ##Tap or flick to remove excess liquid from slides; place in slide rack; add ~4 drops of appropriate probe, cover the section ##Incubate for (at least) 2 hours at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP1 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP1''' to cover each section ##Incubate 30 minutes at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP2 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP2''' to cover each section ##Incubate 15 minutes at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP3 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP3''' to cover each section ##Incubate 30 minutes at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP4 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP4''' to cover each section ##Incubate 15 minutes at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP5 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP5''' to cover each section ##Incubate 30 minutes at RT ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP6 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP6''' to cover each section ##Incubate 15 minutes at RT ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #Signal Detection ##Mix equal volumes of Brown-A and Brown-B (DAB substrate) (e.g. 2 drops each); make ~120 uL DAB per section, mix well 3-5 times ##Tap or flick to remove excess liquid from slides; place ~120 uL DAB onto each section ##Incubate 10 minutes at RT ##Submerge slides in DDI water; agitate 3-5 times; replace with fresh DDI water #Counterstain ##Submerge slides in 50% hematoxylin I solution for 2 minutes at RT; slides will be purple ##Immediately transfer slides back to DDI water; agitate 3-5 times; repeat with fresh DDI water until slides are clear and sections are purple ##Replace DDI water with 0.02% ammonia water; agitate 2-3 times; sections should turn blue ##Submerge slides in DDI water, wash 3-5 times #Dehydrate ##Submerge slides in 70% EtOH in fume hood for 2 minutes with occasional agitation ##Submerge slides in 100% EtOH in fume hood for 2 minutes with occasional agitation ##Submerge slides in fresh 100% EtOH in fume hood for 2 minutes with occasional agitation ##Submerge slides in XYLENE for 5 minutes in fume hood with occasional agitation #Mounting ##Remove slides from xylene and lay flat with sections up in the fume hood ##Mount slides by adding 1-2 drops of cytoseal mounting medium and seal with a coverslip; avoid air bubbles ##Air dry slides for at least 5 minutes #Evaluation ##Evaluate under standard brightfield microscope at 20-40X magnification ##Positive control signal should be visible as punctate dots within cell nucleii at 20-40X magnification ##Negative control may have signal; One dot to every 10 cells is acceptable ##Scoring: [[Image:RNAScope_Scoring_Card.png|600px]] ===Buffer Prep=== #1X Wash Buffer ##Warm 50X wash buffer to 40C for 10-20 minutes before mixing ##Add 2.94L DDI water and 60 mL 50X wash buffer to make 3 L 1X wash buffer ##1X wash buffer may be stored at RT for up to 1 month #50% Hematoxylin I ##Work in the fume hood ##Add 50% Hematoxylin by mixing hematoxylin I and DDI water ##Can be reused up to 1 week #0.02% Ammonia #Add 1.43 mL of 1N ammonium hydroxide to 250 mL DI water; mix well 3-5 times '''Do not reuse following reagents''' #XYLENE #70% and 100% EtOH used in Dehydration during counterstain ==Multiplex Assay (Nuclei)== '''Note''': This is actually the protocol for cultured cells, not nuclei ===Nuclei Prep=== #[[Daniel:Protocols/NuclearAdhesion|Adhere nuclei to slides]] #Rehydration ##Incubate slides in 70% EtOH for 2 minutes ##Incubate slides in 50% EtOH for 2 minutes ##Incubate slides in 1X PBS for 10 minutes ##May want to retrace hydrophobic barrier; can skip #Apply Pretreat 3 ##Remove excess liquid; add 2-4 drops of pretreat 3 ##Incubate for 10 min at RT (in oven) ##Flick to remove excess liquid ##Rinse in fresh 1X PBS; be sure to agitate to clean well ===Multiplex Fluorescent Assay=== #Prepare-Reagents ##Prepare probes by warming to 40C for 10 minutes, then cool to RT ##Briefly spin down C2 and C3 probes to collect the liquid ##Combine C2, C3, and C1 probes by pipetting into a new 1.5mL tube at 1:1:50 ratio ##Prepare 1X Wash Buffer ##Place AMP1-4 FL reagents at RT ##Ensure HybEZ OVEN and Control Tray are at 40C #Hybridize Probe ##Flick to remove excess liquid; add 2-4 drops of probe ##Incubate for 2 hrs at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #AMP1-FL ##Flick to remove excess liquid ##Add 2-4 drops of '''AMP1-FL'''; Incubate for 30 minutes at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #AMP2-FL ##Flick to remove excess liquid ##Add 2-4 drops of '''AMP2-FL'''; Incubate for 15 minutes at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #AMP3-FL ##Flick to remove excess liquid ##Add 2-4 drops of '''AMP3-FL'''; Incubate for 30 minutes at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #AMP4-FL ##Flick to remove excess liquid ##Add 2-4 drops of '''AMP4-FL ALT B'''; Incubate for 15 minutes at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #Counterstain and Mount ##Flick to remove excess liquid; ##Add 2-4 drops '''DAPI'''; Incubate 30 sec at RT ##Remove DAPI and immediate place 1-2 drops of fluorescent mounting medium ##Seal with a coverslip; avoid bubbles #Store in the dark at 4C #Recommended Viewing at 20-40X magnification ===Wavelengths and Microscopy=== '''Color Modules''' [[Image:RNAScopeColorModules.png|500px]] '''Emission/Excitation''' [[Image:RNA_Scope_Excitation_Emission_Spectra.png|500px]] '''Microscope Filters''' [[Image:RNA_Scope_Microscope_Filters.png|600px]] ===RNA Scope Positive Control=== [http://www.acdbio.com/store/rnascoper-3-plex-positive-control-probe-hs.html Positive Control Genes/Probes] [http://www.acdbio.com/control-slides-and-probes Positive Control Genes] *[http://www.genecards.org/cgi-bin/carddisp.pl?gene=POLR2A&keywords=POLR2A POLR2A (C1)] *[http://www.genecards.org/cgi-bin/carddisp.pl?gene=PPIB&keywords=PPIB PPIB (C2)] *[http://www.genecards.org/cgi-bin/carddisp.pl?gene=UBC&keywords=UBC UBC (C3)]
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