Daniel:Notebook/RNAFISH/2014-12-9: Difference between revisions

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>Djacobse
(Created page with "=Cell Line RNA FISH= Back to Calendar ==Dye Labeling== Since I am planning on doing RNA FISH using CUX2 and GAD1, and since I would like to use ...")
 
>Djacobse
 
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#Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
#Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
#Recommend column purification after incubation-[[Daniel:Protocols/CentriSep|Centri-Sep Column]]
#Recommend column purification after incubation-[[Daniel:Protocols/CentriSep|Centri-Sep Column]]
===Labeling Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCC0DA;font-size:12pt" align="center"
| width="96" height="30" | Sample
| width="65" | ng/uL ssDNA
| width="65" | pmol/uL dye
| width="71" | pmol/uL ssDNA
| width="71" | dye:probe
| width="71" | bp:dye
|- style="font-size:12pt"
| height="15"  valign="bottom" | GAD1-488
| align="center" align="center" valign="bottom" | 28.3
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 4.3
| align="center" align="center" valign="bottom" | 1.2
| align="center" align="center" valign="bottom" | 17
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | CUX2-594
| align="center" align="center" valign="bottom" | 21.4
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 3.2
| align="center" align="center" valign="bottom" | 1.2
| align="center" align="center" valign="bottom" | 16
|}
==Hybridization==
'''Sample Matrix'''
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Sample
| width="65" | Cell Line
| width="78" | uL GAD1-488 (ng)
| width="75" | uL CUX2-594 (ng)
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | A1
| align="center" valign="bottom" | U87-MG
| align="center" valign="bottom" | 3 (85)
| align="center" valign="bottom" | 3 (85)
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | B1
| align="center" valign="bottom" | BE2-C
| align="center" valign="bottom" | 4 (86)
| align="center" valign="bottom" | 4 (86)
|}
#Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide
#Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea. 
##Make solutions from absolute ethanol and depc/H20*.
#Dry in oven at 50-65C.
#Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
#Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes. 
#Drop temp to 65C
#Spin briefly.  Do not open the tube when it is at 90C-probe loss can occur. 
#Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
#Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
#Hybridize overnight appropriate temperature
##VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
##important to keep slides in a humid environment
*Prehybridization solution:
**50% deionized formamide
**50% 2xSSC pH 7.0
**Add dextran sulfate to 1%
*Hybridization solution (Arjun Raj)
**2X SSC
**10% Formamide
**10% Dextran Sulfate
**0.02% RNAse-free BSA
**50 μg E.coli tRNA

Latest revision as of 21:20, 9 December 2014

Cell Line RNA FISH[edit]

Back to Calendar

Dye Labeling[edit]

Since I am planning on doing RNA FISH using CUX2 and GAD1, and since I would like to use Alexa 488 and 594, the best dyes I think, I need to do dye labeling. I should be able to use the Alexa dyes, since these are the probes I ordered from IDT.

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 1 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
  8. Recommend column purification after incubation-Centri-Sep Column

Labeling Results[edit]

Sample ng/uL ssDNA pmol/uL dye pmol/uL ssDNA dye:probe bp:dye
GAD1-488 28.3 5 4.3 1.2 17
CUX2-594 21.4 4 3.2 1.2 16

Hybridization[edit]

Sample Matrix

Sample Cell Line uL GAD1-488 (ng) uL CUX2-594 (ng)
A1 U87-MG 3 (85) 3 (85)
B1 BE2-C 4 (86) 4 (86)
  1. Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide
  2. Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
    1. Make solutions from absolute ethanol and depc/H20*.
  3. Dry in oven at 50-65C.
  4. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  5. Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
  6. Drop temp to 65C
  7. Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
  8. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  9. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  10. Hybridize overnight appropriate temperature
    1. VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
    2. important to keep slides in a humid environment
  • Prehybridization solution:
    • 50% deionized formamide
    • 50% 2xSSC pH 7.0
    • Add dextran sulfate to 1%
  • Hybridization solution (Arjun Raj)
    • 2X SSC
    • 10% Formamide
    • 10% Dextran Sulfate
    • 0.02% RNAse-free BSA
    • 50 μg E.coli tRNA