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=RNA FISH-Cell Lines= [[Daniel:Notebook/RNAFISH|Back to Calendar]] ==Hybridization== Today I'm going to perform hybridization on the cell lines I fixed [[Daniel:Notebook/RNAFISH/2014-11-22|Saturday]]. I'll be using RBFOX3 (NeuN) and FOXP2 as my genes of choice. '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | width="65" height="49" | Sample | width="65" | Cell Line | width="81" | uL [[Daniel:Notebook/RNAFISH/2014-10-11|RBFOX3-488]] (ng) | width="79" | uL [[Daniel:Notebook/RNAFISH/2014-10-11|FOXP2-594]] (ng) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | A1 | align="center" valign="bottom" | BE2-C | align="center" valign="bottom" | 2 (110) | align="center" valign="bottom" | 2 (120) |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | B1 | align="center" valign="bottom" | T98-G | align="center" valign="bottom" | 2 (110) | align="center" valign="bottom" | 2 (120) |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | C1 | align="center" valign="bottom" | U-87MG | align="center" valign="bottom" | 2 (110) | align="center" valign="bottom" | 2 (120) |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | D1 | align="center" valign="bottom" | NCI h1975 | align="center" valign="bottom" | 2 (110) | align="center" valign="bottom" | 2 (120) |} #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
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