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=iNGN Bipolar Neurons RNA FISH= *[[Matt:LabNotes/2015-6-8 | First Try]] *[[Matt:LabNotes/2015-6-18 | Second Try]] *[[Matt:LabNotes/2015-6-15 | Probe dye coupling]] *[[Matt:LabNotes/2015-6-30 | Third Try with cooled CCD camera]] **Finally saw some signal using CUX2-Alexa488 48 probes with Sebastian's motor neurons **Camera did not have DAPI filters so did not use DAPI **Only used one probe set per sample *[[Matt:LabNotes/2015-7-1 | Samples are iNGN Bipolar Neuron's from Alex at Harvard]] **Fixed 6/30/2015 and stored in 70% EtOH at 4C for ~9days before RNA FISH **[[Media:INGN-RNAseq-bipolarneurons_Harvard.xlsx | RNA-Seq FPKM data]] *RNA FISH 48 Probes available **CUX2, SLC6A1, ADARB2, SATB2, KIT, SNAP25 ***ADARB2 not in DARTFISH CA12kNov2014_V4 *SNAP25 has highest FPKM value *CUX2 is low-medium expression but use as positive control because it was the only one that worked previously **One sample exact same as last time CUX2-Alexa488 with no DAPI **One sample with odd CUX2-Alexa488 and even CUX2-Alexa594 to check specificity (should colocalize or else non-specific binding) **One sample with CUX2-Alexa488 and SNAP25-Alexa 594 ===Combining CUX2_odd and CUX2_even=== #Oligos already resuspended in 15 uL nuclase-free H2O (1 mM) #Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM) ##Store the remaining oligos at -20C #Add 230 uL nuclase-free, diluting 10:1, for final concentration of 100 uM #*Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower... #*Goal is to get 1ug/ul DNA concentration #Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''measured (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''normalized (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Average''' | align="center" style="background:#f0f0f0;"|'''Stdev''' |- | CUX2_odd 1:1||1251.5||1251.5|||| |- | CUX2_odd 1:5||247||1235|||| |- | CUX2_odd 1:10||117.4||1174||1220||40.8 |- | CUX2_even 1:1||1251.8||1251.8|||| |- | CUX2_even 1:5||235.9||1179.5|||| |- | CUX2_even 1:10||119.1||1191||1207||38.9 |- |} *Added 55ul H2O to each (275ul) to make 330ul of 1000ng/ul ==Dye Coupling== [[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br> [[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] CUX2-488 CUX2_odd-488 CUX2_even-594 SNAP25-594 #Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature #Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer #Denature samples for 5 minutes at 95C, then snap cool using ice box #Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing #*Dye cannot be saved for later use. Use immediately! #Add 8 uL sample to dye tube #Incubate in the dark for 1 hour #Add 10 uL 3M NaOAc and 80 uL 1xTE to sample #[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation #*Use 1X TE Buffer #*No vacufuge #*Disturbed gel bed in CUX2_odd-488 #Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight #Centrifuge at 4 C for 30 minutes (14,000 rpm) #Remove supernatant and add 750 uL chilled 80% EtOH #Centrifuge 5 minutes at 4 C #Dry the pellet in the hood #Resuspend pellet in 12 uL TE #Check dye:probe ratio with Nanodrop #*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) #*Starting DNA mass = 5ul * 1ug/ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe''' | align="center" style="background:#f0f0f0;"|'''ssDNA (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''488 dye (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''594 dye (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''dye:probe ratio''' | align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA start mass (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' |- | CUX2-488||197.2||33.1||0.5||1.09||2.37||5||47.3 |- | CUX2_odd-488||244||40.6||0.6||1.08||2.93||5||58.6 |- | CUX2_even-594||295.9||-0.8||57.3||1.26||3.55||5||71 |- | SNAP25-594||227.4||-0.6||47.5||1.36||2.73||5||54.6 |} ==RNA FISH Procedure== *Sample 1 labeled: "RNAFISH1 7.9.2015" **CUX2_odd-488 and CUX2_even-594 in Sebastian's neurons *Sample 2 labeled: "RNAFISH2 7.9.2015" **CUX2-488 only in Alex's neurons *Sample 3 labeled: "RNAFISH3 7.9.2015" **CUX2_odd-488 and CUX2_even-594 in Alex's neurons *Sample 4 labeled: "RNAFISH4 7.9.2015" **CUX2-488 and SNAP25-594 in Alex's neurons #Prepare 10ml Wash Buffer and let sit at RT #Thaw 500ul aliquot Hybridization Buffer and warm up to 37C #*Aliquots made by Dan #Prepare hybridization reaction #*Hybridization Buffer 100ul #*Probe 5ul for each dye-probe (final volume will be 105ul or 110ul) #Wash the cells with 1ml of PBS (RNase free) 2 times #Add 2ml Wash Buffer and let sit 10min at RT #Aspirate #Add 105ul or 110ul hybridization solution to each sample and incubate overnight at 37C (~22hrs) #Prepare 25ml Wash Buffer #Warm up 16ml Wash Buffer and 8ml Wash Buffer with DAPI to 37C #Rinse with 2ml Wash Buffer #Add 2ml Wash Buffer and incubate 30min at 37C #Replace with 2ml Wash Buffer with DAPI and incubate 30min at 30C #Wash with 2X SSC (warmed to 30C) twice #Add 2X SSC ====Buffer Prep==== *Wash Buffer **20X SSC 5mL **Formamide 5mL **RNase free H2O 40ml *Wash Buffer with DAPI **Wash Buffer 10ml **DAPI (100ug/ml) 50ul ***Stored in brown opaque tube in 4C *Hybridization Buffer **RNAse free water 5.3 mL **SSC 20X 1 mL **Dextran sulfate 2 mL **Formamide 1 mL **E coli tRNA 500 uL **RVC 200 mM (warm to 37) 100 uL **BSA 50 mg/mL 40 uL ==Imaging Results== *RNAFISH1: Could see faint signal in 488 channel but nothing specific in 594 channel **Fainter signal in 488 compared to [[Matt:LabNotes/2015-6-30 | Third Try w/ cooled CCD camera]] is expected since only 24probes versus 48probes **Failure to see bright distinct spots in 594 channel but instead see bright regions of cells that correspond to dense dark areas in phase channel ***My guess is that dye-probes or maybe free dyes got trapped? *RNAFISH2: Few spots in 488 representing CUX2 transcripts in Alex's neurons **I believe these neurons express less CUX2 than Sebastian's (based on FPKM~2 in Alex's RNA-Seq data and Sebastian claiming CUX2 is "highly expressed" in his neurons) *RNAFISH3: Results similar to RNAFISH1 *RNAFISH4: CUX2 spots are detected but SNAP25 (which should have high expression) is not detected **The Alexa594 channel (SNAP25) shows the same bright regions of cells as the other dishes with 594 channel ***This suggests the bright regions is not due to probe sequence but property of the dye *Conclusion: **Only CUX2-488 probes have worked and there is less CUX2 in Alex's iNGN bipolar neurons than Sebastian's motor neurons **CUX2-594 and SNAP25-594 both exhibited similar peculiarities and suggests the issue lies with Alexa594 dye or Alexa594 dye-coupling (See dye:probe ratio from dye-coupling results) **Need to call LifeTech ARES or Biosearch Stellaris customer support for advice on dye-coupling and bright regions **Next experiment should try switching probe and dye combinations (CUX2-488 and SNAP25-594) ===Images=== ====RNAFISH1==== *CUX2-488 [[File:4_w2Alexa488-Matt.jpg]] *CUX2-594 [[File:4_w1Alexa594-Matt.jpg]] ====RNAFISH2==== *CUX2-488 [[File:RNAFISH2_20150709_4_w2Alexa488-Matt.jpg]] ====RNAFISH3==== *CUX2-488 [[File:RNAFISH3_20150709_8_w2Alexa488-Matt.jpg]] *CUX2-594 [[File:RNAFISH3_20150709_8_w1Alexa594-Matt.jpg]] *Phase [[File:RNAFISH3_20150709_8_w4Phase-WPZ.jpg]] ====RNAFISH4==== *CUX2-488 [[File:RNAFISH4_20150709_9_w2Alexa488-Matt.jpg]] *SNAP25-594 [[File:RNAFISH4_20150709_9_w1Alexa594-Matt.jpg]] *Phase [[File:RNAFISH4_20150709_9_w4Phase-WPZ.jpg]] ====Settings==== *7/10/2015 *Dr. Bern's Zeiss microscope SERF 295 **Light source: X-Cite 120LED **Objective: 100X NA=1.4 oil **Camera: QuantEM 512SC EMCCD Camera **Cubes: Color (Ex filter - Dichroic mirror - Em filter) ***DAPI - (350/50 - 400 - 460/50) ****5% intensity 25msec exposure ***FITC (Alexa488)- (480/30 - 510 - 535/40) ****100% intensity 5sec exposure ***dsRED (Alexa594) - (545/30 - 570 - 628/52) ****20% intensity 150msec exposure ===StarSearch===
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