ࡱ> []Z[@ 0bjbj44 "2ViVi|dddd$$|G2Xlj8%G'G'G'G'G'G'G$HRNKKG9KGG.%G%G9CD G ˳d"}D&%GG0GDfKKL G&4Z"K G"KGKG||d  ||LCM Protocol Slide Preparation: For optimal RNA recovery use frozen tissues Paraffin-embedded tissue is not appropriate for gene expression analysis due to the difficulties in isolating RNA Use Rnase-free technique throughout Cut 6-8um sections of tissue and apply to slide Section multiple slides at once Recommended type of LCM caps: CapSure( HS LCM Caps (produced by Arcturus cat# LCM0201  HYPERLINK "http://www.arctur.com/home/system_index.htm" http://www.arctur.com/home/system_index.htm) Optimal for smaller number of cells Only 10 ul of extraction volume needed (when using Arcturus extraction methods) Stand-off rails on the cap keep the transfer film out of contact with tissue Staining Techniques: Stain according to your protocol (very specific for the cells you are interested in) Note: do not fix slides in acetone Keep in mind that staining protocols should not be too time intensive (RNA will start to degrade) To Dehydrate: 30 sec DEPC-H2O 30 sec 70% EtOH 1 min 100% EtOH 1 min 100% EtOH 1 min 100% EtOH 1 min 100% EtOH 5 min Xylene 25-30 min in the hood The following are commercially available kits to aid in the staining process: HistoGene( Frozen Section Staining Kit by Arcturus ( HYPERLINK "http://www.arctur.com/products/histo_stain.htm" http://www.arctur.com/products/histo_stain.htm) It is a fast penetrating stain that provides good contrast by differential staining of nuclei (purple) and cytoplasm (light pink). Retrieve high quality RNA from stained samples Protocol available at  HYPERLINK "http://www.arctur.com/images/pdf/histogene_user_guide.pdf" http://www.arctur.com/images/pdf/histogene_user_guide.pdf HistoGene"! LCM Immunofluorescence Staining Kits by Arcturus ( HYPERLINK "http://www.arctur.com/products/histo_stain.htm" http://www.arctur.com/products/histo_immuno_simple_process.htm) Universal Immuno-fluorescence using biotinylated primary antibody Protocol available at  HYPERLINK "http://www.arctur.com/images/pdf/histogene_immuno_user_guide_A.pdf" http://www.arctur.com/images/pdf/histogene_immuno_user_guide_A.pdf Complete LCM protocol within 2 hours Do not refreeze a stained tissue sectiononce stained, LCM must be performed Partial RNA degradation may occur Exposure to humidity, air, moisture may decrease RNA quality LCM protocol (Using the Arcturus Applied Genomics (AAG) PixCell( IIe, LCM): (please contact Arcturus for a more detailed description) Make sure that the joystick is perpendicular to the table Place slide on microscope platform, apply vacuum Load cap onto swingarm and lower onto tissue section Ensure that the film is able to contact the glass Turn laser key on, push laser enable button (located on controller) Adjust laser diameter to small (7.5um) and focus the laser to a bright, well-defined spot Adjust laser power and duration settings to capture cells (see below). Pulse laser at target cells Remove cap with adhered target cells Proceed with RNA extraction techniques The amount of RNA you are able to isolate is VERY tissue-dependent LASER SPOT SIZE POWER DURATION Small (7.5um) 65-75mW 1000 1500us Medium (15um) 35-45mW 2.5 3.0ms Large (30um) 45-55mW 6.0 7.0ms RNA Extraction Techniques PicoPure( RNA Isolation Kit by Arcturus (cat # KIT0202 and KIT0204  HYPERLINK "http://www.arctur.com/products/picorna_deliver.htm" http://www.arctur.com/products/picorna_deliver.htm) For use with as little as 10 cells Protocol located at  HYPERLINK "http://www.arctur.com/images/pdf/picopure_rna_user%20guide.pdf" http://www.arctur.com/images/pdf/picopure_rna_user%20guide.pdf RiboAmp RNA Amplification Kit by Arcturus (cat # KIT0201 and KIT0203  HYPERLINK "http://www.arctur.com/products/riboamp_main.htm" http://www.arctur.com/products/riboamp_main.htm) 1-4 ng yields 30-50 ug Protocol located at  HYPERLINK "http://www.arctur.com/images/pdf/riboamp_user_guide.pdf" http://www.arctur.com/images/pdf/riboamp_user_guide.pdf Absolutely RNA Microprep Kit ( HYPERLINK "http://www.stratagene.com/displayProduct.asp?productId=530" http://www.stratagene.com/displayProduct.asp?productId=530) produced by Stratagene Designed to isolate total RNA from samples containing up to 5 x 105 cells Protocol located at  HYPERLINK "http://www.stratagene.com/manuals/400805.pdf" http://www.stratagene.com/manuals/400805.pdf Note: Do not use Trizol( has been found to degrade the LCM transfer film All information pertaining to PixCell( IIe LCM, RiboAmp, PicoPure, HistoGene, PixCell, CapSure was taken from the Arcturus website located at  HYPERLINK "http://www.arctur.com/home/system_index.htm" http://www.arctur.com/home/system_index.htm. RiboAmp, PicoPure, HistoGene, PixCell, CapSure, ExtracSure and MiraCol are trademarks owned by Arcturus. All information pertaining to the Absolutely RNA Microprep Kit was taken  !8 T _ ` . / 1 ^z|:;*+=>J jh^'>*jh^'Ujh^'Uh^'B* aJphfjVh^'U h^'aJj%h^'U h^'0Jjh^'Ujh^'U jh^' h^'>*h^' h^'CJ6 !"P 7 8 V X  n ' J !!!!!H!Ճ!H!H!H!!!!ޅ!!!!!!!H!!!!H!H!H & F & F & F & F & F0  ' = > 1 =,QJ!H!H!H!H!H!!!!ޅ!!!!!Ճ!!!!!!!!Q!v:!! & F$a$ & F & F^ & FJNO IQV^_ABCz{ܹ䝪jH h^'Uh^'0JaJj h^'UaJjh^'UaJ h^'H* h^'5\jh^'U h^'0Jjeh^'Ujh^'U h^'aJ jh^' h^'>*h^' h^'CJ2%W<X} 1UyzQt!!!!!!!!!!!!!!!!!!!ޅ!!!!  & F ^ & F  & F^^ & F  & F|:OPQR0000!!!b0!!!!!!!!^!v!^!^!^!!h^h8^8 & F  & F & F 8^8` !:|}QpwxϴϢyjyZyjh^'CJOJQJU^J jh^'CJOJQJ^Jh^'CJOJQJ^Jh^'CJOJQJ^JaJ jh^'h^'0JaJ"j h^'B*UaJphjh^'B*UaJphh^'B*H*aJphh^'B*aJph h^'0Jj h^'Ujh^'Uh^'h^'CJH*aJGH0'0(0P0Q0R0l0m0000³±ž™h^' h^'>*%jLh^'CJOJQJU^JUh^'CJH*OJQJ^JaJh^'CJOJQJ^Jh^'0JCJOJQJ^Jjh^'CJOJQJU^J%j'h^'CJOJQJU^Jfrom the Stratagene website located at  HYPERLINK "http://www.stratagene.com/" http://www.stratagene.com/. 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