Daniel:Protocols/RNAScope: Difference between revisions
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##Submerge in fresh 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 | #AMP6 | ||
##Tap or flick to remove excess liquid from slides; add ~4 drops of ''' | ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP6''' to cover each section | ||
##Incubate 15 minutes at RT | ##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 | ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down | ||
Line 177: | Line 177: | ||
##Seal with a coverslip; avoid bubbles | ##Seal with a coverslip; avoid bubbles | ||
#Store in the dark at 4C | #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)] |
Latest revision as of 18:31, 28 October 2015
RNA Scope[edit]
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[edit]
- Fresh Frozen Sample Prep User Manual
- Multiplex Fluorescent Assay User Manual
- BROWN Detection Kit Manual
- Fresh Frozen Tissue Prep Quick Guide
- Cultured Cells Manual
Chromogenic Staining Protocol[edit]
Tissue Prep (~30 minutes)[edit]
- 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[edit]
- 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:
File:RNAScope Scoring Card.png
Buffer Prep[edit]
- 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)[edit]
Note: This is actually the protocol for cultured cells, not nuclei
Nuclei Prep[edit]
- 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[edit]
- 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[edit]
Color Modules
Emission/Excitation
File:RNA Scope Excitation Emission Spectra.png
Microscope Filters
File:RNA Scope Microscope Filters.png