Daniel:Notebook/RNAFISH/2014-8-31: Difference between revisions
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#Incubate in the dark for 1 hour | #Incubate in the dark for 1 hour | ||
#Add 5 uL 3M NaOAc and 40 uL nfH20 to sample | #Add 5 uL 3M NaOAc and 40 uL nfH20 to sample | ||
# | #[[Daniel:Protocols/CentriSep|Centri Sep]] purification after incubation | ||
===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="85" height="31" | Sample | |||
| width="65" | ng/uL ssDNA | |||
| width="64" | pmol/uL dye | |||
| width="65" | pmol/uL ssDNA | |||
| width="65" | dye:probe | |||
| width="65" | bp:dye | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | GAD1-488 | |||
| align="center" align="center" valign="bottom" | 55.7 | |||
| align="center" align="center" valign="bottom" | 9.2 | |||
| align="center" align="center" valign="bottom" | 8.4 | |||
| align="center" align="center" valign="bottom" | 1.1 | |||
| align="center" align="center" valign="bottom" | 18 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | FOXP2-546 | |||
| align="center" align="center" valign="bottom" | 67.7 | |||
| align="center" align="center" valign="bottom" | 13.7 | |||
| align="center" align="center" valign="bottom" | 10.3 | |||
| align="center" align="center" valign="bottom" | 1.3 | |||
| align="center" align="center" valign="bottom" | 15 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | RBFOX3-594 | |||
| align="center" align="center" valign="bottom" | 68.3 | |||
| align="center" align="center" valign="bottom" | 13.8 | |||
| align="center" align="center" valign="bottom" | 10.3 | |||
| align="center" align="center" valign="bottom" | 1.3 | |||
| align="center" align="center" valign="bottom" | 15 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | CUX2-647 | |||
| align="center" align="center" valign="bottom" | 59.8 | |||
| align="center" align="center" valign="bottom" | 12.3 | |||
| align="center" align="center" valign="bottom" | 9.1 | |||
| align="center" align="center" valign="bottom" | 1.4 | |||
| align="center" align="center" valign="bottom" | 15 | |||
|} | |||
Great labeling results, and each sample has ~50 uL to work with. | |||
==Pretreatment== | ==Pretreatment== | ||
'''Sample Matrix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#FCD5B4;font-size:12pt" | |||
| align="center" width="85" height="30" valign="bottom" | | |||
| width="83" align="center" valign="bottom" | uL GAD1-488 (pmol) | |||
| width="85" align="center" valign="bottom" | uL FOXP2-546 (pmol) | |||
| width="80" align="center" valign="bottom" | uL RBFOX3-594 (pmol) | |||
| width="80" align="center" valign="bottom" | uL CUX2-647 (pmol) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample A | |||
| align="center" | 25 (211) | |||
| align="center" align="center" | 0 | |||
| align="center" | 25 (258.7) | |||
| align="center" align="center" | 0 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample B | |||
| align="center" align="center" | 0 | |||
| align="center" | 25 (256.4) | |||
| align="center" align="center" | 0 | |||
| align="center" | 25 (226.5) | |||
|} | |||
Sample A is the sample closer to the label, sample B is the sample further from the label. | |||
#Remove slides from 4C, let them thaw and air-dry at RT for 30–60 min | #Remove slides from 4C, let them thaw and air-dry at RT for 30–60 min | ||
Line 62: | Line 137: | ||
#Remove the Coplin jar from the water bath and let it cool down at RT | #Remove the Coplin jar from the water bath and let it cool down at RT | ||
#Incubate the slides in 2× SSC for 5 min | #Incubate the slides in 2× SSC for 5 min | ||
#Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h | #Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h | ||
==Hybridization== | ==Hybridization== | ||
Line 78: | Line 153: | ||
#Let the rubber cement dry at RT | #Let the rubber cement dry at RT | ||
##From this step, protect slide from light | ##From this step, protect slide from light | ||
#Place slide on a hot-block (or water bath) with temperature 45°C for | #Place slide on a hot-block (or water bath) with temperature 45°C for 2 h in order to allow infiltration of the section with the probe | ||
#Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA | #Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA | ||
#Hybridize slide for | #Hybridize slide for 3 days at 37°C in a waterbath or temperature controlled oven; [[Daniel:Notebook/RNAFISH/2014-9-3|Continued 9/3/2014]] | ||
[[Category:RNAFISH]] [[Category:Hybridization]] [[Category:20140829]] [[Category:ARES Label]] |
Latest revision as of 17:10, 24 September 2014
Trial Run (Started 08/29/2014)[edit]
Dye Labeling[edit]
Sample Matrix
Sample | GAD1 | FOXP2 | RBFOX3 | CUX2 |
Dye | Alexa-488 | Alexa-546 | Alexa-594 | Alexa-647 |
ng/uL | 512 | 558 | 568 | 604 |
uL added | 10 | 10 | 10 | 10 |
ug added | 5.1 | 5.6 | 5.7 | 6.0 |
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 5 uL DNA 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 solvent; vortex ≥10 seconds
- dye cannot be saved for later use. Use immediately!
- Add 2 uL dye to sample
- Incubate in the dark for 1 hour
- Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
- Centri Sep purification after incubation
Labeling Results[edit]
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye:probe | bp:dye |
GAD1-488 | 55.7 | 9.2 | 8.4 | 1.1 | 18 |
FOXP2-546 | 67.7 | 13.7 | 10.3 | 1.3 | 15 |
RBFOX3-594 | 68.3 | 13.8 | 10.3 | 1.3 | 15 |
CUX2-647 | 59.8 | 12.3 | 9.1 | 1.4 | 15 |
Great labeling results, and each sample has ~50 uL to work with.
Pretreatment[edit]
Sample Matrix
uL GAD1-488 (pmol) | uL FOXP2-546 (pmol) | uL RBFOX3-594 (pmol) | uL CUX2-647 (pmol) | |
Sample A | 25 (211) | 0 | 25 (258.7) | 0 |
Sample B | 0 | 25 (256.4) | 0 | 25 (226.5) |
Sample A is the sample closer to the label, sample B is the sample further from the label.
- Remove slides from 4C, let them thaw and air-dry at RT for 30–60 min
- Re-hydrate sections in 10 mM sodium citrate buffer for 5 min
- Transfer slides into a Coplin jar with prewarmed (80°C) 10 mM sodium citrate buffer in a water bath and incubate there for 5–25 min
- Remove the Coplin jar from the water bath and let it cool down at RT
- Incubate the slides in 2× SSC for 5 min
- Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h
Hybridization[edit]
- Remove slide from the formamide and wipe the liquid from the back of the slide and from the face side of the slide
- Avoid marked section area
- Prepare glass hybridization chambers as follows:
- Roll a small piece of mounting putty (Blu Tack) into a snake.
- Wrap the mounting putty around tissue section, covering 3 sides
- Size of the chamber depends on a section size: chamber should fit to the section size as closely as possible in order to save DNA probe
- Place coverslip over putty, press gently to seal the coverslip to the putty
- Load the probe under chamber from its open side so that probe fills in the whole space above the section
- Seal the chamber with rubber cement
- It is important that rubber cement covers the entire chamber periphery or even the whole chamber
- Let the rubber cement dry at RT
- From this step, protect slide from light
- Place slide on a hot-block (or water bath) with temperature 45°C for 2 h in order to allow infiltration of the section with the probe
- Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA
- Hybridize slide for 3 days at 37°C in a waterbath or temperature controlled oven; Continued 9/3/2014