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=RNA FISH Based Cell Sorting= [[Daniel Jacobsen|Back to Main]] [http://www.nature.com/nmeth/journal/v11/n5/pdf/nmeth.2910.pdf Original paper by Klemm et al] The goal of this protocol is to be able to use RNA FISH when doing cell sorting, which can be a potentially more valuable way of cell sorting than using antibody staining, as it provides greater discrimination prospects. ==Protocol== NOTE: The original protocol (from the paper) is rather vague and several steps may require troubleshooting if my guesses as to their meaning are not correct. ===RNA FISH=== #Fix cells in 4% paraformaldehyde for 5 min #Centrifuge cells at 1000g for 5 minutes #Wash cells with 70% EtOH #Ethanol permeabilization- 70% EtOH overnight at 4C #Resuspend cells in RPHB with labeled probes (0.5-1ng/uL) ##Incubate for 12 hrs at 30C #Add equal volume of wash buffer and mix thoroughly #Pellet by centrifugation (1000g for 5 min) #Resuspend cells in wash buffer for 30 minutes at 30C #Pellet by centrifugation and resuspend cells in wash buffer for 30 minutes at 30C #Pellet cells and resuspend in flow buffer at 4C in preparation for sorting ===Flow Cytometry, FACS, and RNA Extraction=== #Sort by FACS into 4C sorting buffer #Cell size was controlled by costaining with Hoeschst 33342 and selecting for diploid DNA content #Pellet FACS sorted cells using 1000g for 5 minutes and resuspend in reverse cross-linking buffer at 50C for 1 hr #Isolate total RNA via phenol-chloroform or other ===Buffers=== *'''RPHB''' **300 mM NaCl **30 mM sodium citrate **2.1M ammonium sulfate **10 mM EDTA **1 mg/mL E. coli tRNA **500 ug/mL BSA **25% v/v formamide **pH 5.2 *'''Wash Buffer''' **25% v/v formamide **2X SSC buffer *'''Flow Buffer''' **2X SSC *'''Sorting Buffer''' **200mM NaCl **20 mM sodium citrate **1.5M ammonium sulfate **5 mM EDTA **2X SSC **pH 5.2 *'''Reverse Cross-Linking Buffer''' **100mM NaCl **10mM Tris **1 mM EDTA **0.5% SDS (v/v) **500 ug/mL proteinase K ==Buffer Prep== ===RPHB=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt" align="center" | width="112" height="37" | Reagent | width="78" | Stock Concentration | width="77" | Final Concentration | width="65" | Dilution | width="180" | Volume in 10 mL |- style="font-size:12pt" | height="15" valign="bottom" | SSC Buffer | align="center" valign="bottom" | 20X | align="center" valign="bottom" | 2X | align="center" align="center" valign="bottom" | 1:10 | align="center" valign="bottom" | 1 mL |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Ammonium Sulfate | align="center" valign="bottom" | NA, powder | align="center" valign="bottom" | 2.1 M | align="center" valign="bottom" | NA | align="center" valign="bottom" | 2.775g |- style="font-size:12pt" | height="15" valign="bottom" | EDTA | align="center" valign="bottom" | 0.5 M | align="center" valign="bottom" | 10 mM | align="center" align="center" valign="bottom" | 1:50 | align="center" valign="bottom" | 200 uL |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | E. coli tRNA | align="center" valign="bottom" | 20 mg/mL | align="center" valign="bottom" | 1 mg/mL | align="center" align="center" valign="bottom" | 1:20 | align="center" valign="bottom" | 500 uL |- style="font-size:12pt" | height="15" valign="bottom" | BSA | align="center" valign="bottom" | 50 mg/mL | align="center" valign="bottom" | 500 ug/mL | align="center" valign="bottom" | 1:100 | align="center" valign="bottom" | 100 uL |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Formamide | align="center" align="center" valign="bottom" | 100% | align="center" align="center" valign="bottom" | 25% | align="center" align="center" valign="bottom" | 1:04 | align="center" valign="bottom" | 2.5 mL |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | - | align="center" valign="bottom" | - | align="center" valign="bottom" | - | align="center" valign="bottom" | Fill to 10mL (~5 mL) |} ===Sorting Buffer=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt" align="center" | width="112" height="30" | Reagent | width="78" | Stock Concentration | width="77" | Final Concentration | width="65" | Dilution | width="180" | Volume in 10 mL |- style="font-size:12pt" valign="bottom" | height="15" | SSC Buffer | 20X | 2X | align="center" | 1:10 | 1 mL |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | Ammonium Sulfate | NA, powder | 1.5 M | NA | 1.982 g |- style="font-size:12pt" valign="bottom" | height="15" | EDTA | 0.5 M | 5 mM | 1:100 | 100 uL |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | nfH2O | - | - | - | Fill to 10mL (~9 mL) |} ===Reverse Cross-Linking Buffer=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#D8E4BC;font-size:12pt" align="center" | width="112" height="30" | Reagent | width="78" | Stock Concentration | width="77" | Final Concentration | width="65" | Dilution | width="180" | Volume in 10 mL |- style="font-size:12pt" valign="bottom" | height="15" | NaCl | NA, powder | 100 mM | - | 58.4 mg |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | Tris | 1 M | 10 mM | 1:100 | 100 uL |- style="font-size:12pt" valign="bottom" | height="15" | EDTA | 0.5 M | 1 mM EDTA | 1:500 | 20 uL |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | SDS | 10% w/v | 0.5% (w/v) | 1:20 | 500 uL |- style="font-size:12pt" valign="bottom" | height="15" | Proteinase K | 20 mg/mL | 500 ug/mL | 1:40 | 250 uL |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | nfH2O | - | - | - | Fill to 10 mL (~9 mL) |}
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