View RNA
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1. Cy5 Ex 644 nm Em 669 nm Cy7 Ex 740 nm Em 764 nm and DAPI Ex 358 nm Em 461 nm Hybridization of probe sets for FFPE tissue sections Probe sets were diluted 1 50 in prewarmed Hyb A buffer and 200 uL was added to each tissue section which was incubated for 3 hours at 40 C Following this step slides were washed by submersion and agitation for 2 minutes in wash buffer twice Slides were removed from the wash buffer and 200 uL of PreAmp 1 solution was added to the tissue section and incubated at 40 C for 25 minutes Following three washes with wash buffer 200 uL of Amp 1 reagent was added to the tissue section and the slides were incubated at 40 C for 15 minutes Again slides were washed three times in wash buffer and then incubated with Label Probe AP reagent for 40 C for 15 minutes After three washes with wash buffer 200 uL of the AP enhancer solution was added to the section and incubated at room temperature for 5 to 10 minutes Fast Red solution was prepared by dissolving one tablet in 5 mL of naphthol buffer and 200 uL was added to the tissue section which was incubated at 40 C for 30 minutes in the dark Slides were then submerged in 1x PBS in order to rinse off the Fast Red substrate To fix slides were incubated in 4 percent formaldehyde for 5 minutes at room temperature before being washed in 1x PBS to eliminate traces of formaldehyde Slides were counterstained with Gill s hematoxylin American Master Tech Scientific USA befor2. HER2 gene were expressed as well as diploid copies of the housekeeper gene for IL 8 However in the SKBR3 breast cancer cell line multiple copies of HER2 were apparent while copies of the IL 8 gene remained at normal levels Figure 3A This was also observed at the MRNA level Figure 3B Figure 3 DNA 3A and mRNA 3B expression of HER2 in HeLa and SKBR3 cells Her2C a probe directed to the unprocessed intron region of HER2 served as a negative control For DNA detection cells were first treated with RNAse then DNA was denatured prior to jn situ hybridization Nuclei were counterstained with DAPI Figure 3A Figure 3B HeLa SKBR3 Her 2 18S DAPI HER2 IL8 DAPI a lt Q N co O Ra D X This assay has also been used in FFPE sections which are notoriously difficult samples due to the fixation processes involved in preserving them We examined the expression of the HPRT gene in FFPE rat kidney tissue sections using bDNA technology Results demonstrated that individual copies of HPRT mRNA were visible in rat kidney tissue sections Figure 4 To compare sensitivity between the use of radiolabeled probes and our reported method IGFBP 3 mRNA expression was visualized in human liver sections using either S UTP probe or probes designed for use with the bDNA method IGFBP 3 mRNA expression was apparent in liver Kupffer cells using both methods However radiolabeled probes required exposure to X ray film for three w
3. KRG White Paper Affymetrix QuantiGene ViewRNA Assay Simple robust and sensitive visualization of mRNA for in situ analysis Abstract The method of in situ hybridization is an invaluable technique that allows researchers to visualize their gene of interest in target cells and tissues However this technique is time and labor intensive and often limited by lack of sensitivity Here we discuss a new method the QuantiGene ViewRNA Assay which can detect single copies of the target gene in cultured cells The technique can also be applied to formalin fixed paraffin embedded FFPE tissue sections With a combination of greater sensitivity and shortened workflow the QuantiGene ViewRNA Assay markedly improves on currently available in situ hybridization methods Introduction Branched DNA bDNA detection is a clinically proven technology that quantitatively measures gene expression The bDNA assay is a sandwich nucleic acid hybridization method that uses bDNA molecules to amplify signal from captured target RNA Amplification of the signal to enhance assay sensitivity eliminates the need to amplify target RNA as required in traditional PCR based gene expression techniques Furthermore DDNA assays measure RNA directly from the sample source without RNA purification or enzymatic manipulation thereby avoiding inefficiencies and variability introduced by errors inherent to these processes DDNA assays have been available in the clinic
4. Nuclei were counterstained with DAPI Page 1 of 6 This technology enables transcriptional profiling of individual cells within a population and has broad applicability in research areas including biomarker validation as well as in vitro and in vivo quantitation of RNAi knockdown The assay is amenable to automation and the simple assay workflow is suitable for high throughput applications such as phenotypic or reporter gene screening This method can also be used to detect individual RNA targets in FFPE tissue sections Methods A schematic diagram of the workflow is represented in Figure 2A in cultured cells and Figure 2B in FFPE tissue sections Unless otherwise noted reagents for cultured cells referred to in the workflow used are provided in the QuantiGene ViewRNA Plate Based Assay and Signal Amplification Kits For more information about the components provided in the assay please refer to the User Manual QuantiGene ViewRNA Plate Based Assay available at www panomics com Reagents for FFPE tissue sections referred to in the workflow used are provided in the QuantiGene ViewRNA FFPE Assay Kit and Chromogenic Signal Amplification Kit QuantiGene ViewRNA probe sets are ordered separately For more information about the components provided in the assay please refer to the User Manual QuantiGene ViewRNA for FFPE Samples available at www panomics com Figure 2 Workflow of the in situ hybridization assay based on bDNA technology fo
5. al market for more than a decade forming the basis of the FDA approved clinical diagnostic VERSANT 3 0 assays for viral load in human immunodeficiency virus HIV hepatitis B virus and hepatitis C virus infections commercially offered by Siemens This technique is also offered in the preclinical and life sciences markets in a single plex and multiplex assay setup known as QuantiGene 2 0 and QuantiGene Plex 2 0 Assays offered by Affymetrix Due to its system wide accuracy precision and ease of use DDNA is a superior method of analyzing gene expression in a variety of sample types The technique of in situ hybridization is used to visualize the localization of DNA or RNA within cells and tissues However this method has often been limited by low sensitivity and a complicated workflow as well as the inability to perform multiplex analysis The QuantiGene ViewRNA Assay contains a novel mRNA in situ hybridization solution based on patent pending probe set design and bDNA technology The combination of probe design and signal amplification results in an assay that offers single copy MRNA sensitivity in single cells in a multiplex assay format Currently up to four RNA targets can be visualized at a single transcript level within individual cells using this method Figure 1 Figure 1 Expression of four housekeeping genes in HeLa cells using the bDNA jin situ hybridization method RPLO green PPIB yellow HPRT aqua and B actin red
6. bated at 60 C for 30 minutes in a ThermoBrite station Abbott Molecular USA to increase tissue attachment to the slide This was followed by submerging the slides in 10 percent formaldehyde for 1 hour at room temperature Slides were washed briefly in 1x PBS twice before being air dried For deparaffinization slides were incubated at 80 C for 3 minutes followed immediately by submersion in 200 mL of Histo Clear reagent National Diagnostics USA for 10 minutes at room temperature with frequent agitation Residual Histo Clear reagent was removed by Page 3 of 6 agitating slides in 95 percent ethanol twice Slides were allowed to dry at room temperature for 5 minutes before a hydrophobic barrier was drawn around the tissue section using an ImmEdge Hydrophobic Pen Vector Laboratories USA Once the barrier had dried slides were placed into boiling pretreatment solution and incubated for between 5 and 20 minutes depending on tissue type After pretreatment slides were submerged in distilled water twice Protease solution was prepared by diluting protease 1 100 in 1x PBS and warmed to 40 C Slides were also prewarmed to 40 C with 200 uL of 1x PBS covering the tissue section to avoid dehydration Once warmed the 1x PBS solution was decanted from the section and 200 uL of the prewarmed protease solution was added The slides were incubated at 40 C for 10 to 40 minutes depending on tissue type Following this step the protease solution
7. e being mounted using Dako Ultramount medium Dako USA Results were viewed under a bright field microscope Results and discussion In the past in situ hybridization has been limited by sensitivity and a complicated workflow To enhance sensitivity probes were often radiolabeled and exposed to film for several weeks before results were visualized Despite the time intensive procedure detection of MRNA was still limited to approximately 20 copies per cell We report a novel method for visualizing localization of mRNA in cultured cells and FFPE sections Specificity of the assay was achieved by hybridizing multiple double Z structured oligonucleotide pairs along a target region Figure 2A and 2B By using this method of probe design sequences with more than 90 percent homology can still be readily distinguished Furthermore the double Z structure incorporated into the oligonucleotide pair design is critical to the assay as each oligonucleotide pair Page 4 of 6 forms the base of the signal amplification By subsequent addition of the preamplifier amplifier and label probe the signal is amplified 400 fold per each oligonucleotide pair To verify that the assay is sensitive enough to detect single copy transcript in a cell probe sets were designed against HER2 DNA so that copy number could be quantified HER2 is a gene that is overexpressed in certain types of aggressive breast cancers In HeLa cells normal diploid copies of the
8. eeks before results could be visualized This was in contrast to the b DDNA method where IGFBP 3 expression was apparent after a 30 minute chromogenic substrate reaction Figure 5 page 6 Figure 4 Expression of HPRT in FFPE ray kidney B itn BP s Rite o tee S my a tissue HPRT mRNA red nuclei were os P po a hae gt counterstained using hemotoxylin 1 blue E 7s ERa iao OP SE r oe 6 ni lt z ae pie e i j 2 Ss A ne x u i Os E ae C a X Ce S GO nie po on lt S P Fe eS a Yi pt Me ee es y amp A fas es t ane p Ss gt go Coe r 3 Pg i f A a E m m i T 3 69 gt be t e cre eet a9 y lt e Pay w i TF v A 2 On ie P hp Ca a AA iG ate Coo p amp a O lt wu d S 4 oe 2 gt C J oe E A _ Page 5 of 6 Y gt s Ad a amp b gt t gt E T A a k k J ww Ji 4 gt Figure 5 IGFBP 3 mRNA expression in FFPE human liver tissue samples detected using radioactivity and QuantiGene ViewRNA Assay In situ by radiolabeled probes In situ by branched bDNA 4 a lt E J 9 4 gt ra an ae Taken together these results highlight the specificity and sensitivity of the DDNA method for in situ hybridization Furthermore this method has considerably improved the workflow such that the time to obtain results has been significantly reduced compared to traditional methods Please visit www panomics com t
9. nce microscope or automated imaging platform Page 2 of 6 Figure 2B Z Target mRNA specific probe sets z Z Zz zZz y A Z zZ e Z k PreAmp1 r Amp1 OLP AP om FFPE tissue section Target mRNA Step 1 Prepare sample Step 2 Hybridize probe sets Step 3 Amplify signal Step 4 Image Step 1 Prepare sample FFPE tissue samples fixed for 16 to 24 hours at room temperature RT are sectioned to 4 6 um thickness and attached to a positively charged slide FFPE tissue sections are treated with a pretreatment solution followed by protease digestion to allow target accessibility Step 2 Hybridize probe sets A gene specific probe set hybridizes to the target MRNA For clarity only single oligonucleotide pairs are shown However a typical probe set contains 10 or more oligonucleotide pairs Step 3 Amplify signal A Preamplifier PreAmp molecule hybridizes to each pair of oligonucleotides then multiple Amplifier Amp molecules hybridize to each PreAmp Finally multiple Label Probe oligonucleotides conjugated to alkaline phosphatase LP AP hybridize to each Amp Following the addition of the Fast Red Substrate alkaline phosphatase breaks down the substrate to form a precipitate punctated dots that indicates the presence of the target RNA molecule Step 4 Image Target mRNA is visualized using standard bright field microscopy and cell nuclei can be identified by the hematoxylin counterstain Sample preparation f
10. o learn more about the QuantiGene ViewRNA Assay References 1 Canales R D et a Evaluation of DNA microarray results with quantitative gene expression platforms Nature Biotechnology 24 9 1115 1122 2006 2 Zhao J et al Secreted antibody granzyme B fusion protein stimulates selective killing of HER2 overexpressing tumor cells Journal of Biological Chemistry 279 20 21343 21348 2004 3 Yu C J et al Selective proapoptotic activity of a secreted recombinant antibody AIF fusion protein in carcinomas overexpressing HER2 Gene Therapy 13 4 313 320 2006 Affymetrix Inc 1 888 362 2447 Affymetrix UK Ltd 44 0 1628 552550 Affymetrix Japan K K 81 0 3 6430 4020 Panomics Products 1 877 PANOMICS www panomics com USB Products 1 800 321 9322 www usb affymetrix com www affymetrix com Please visit our website for international distributor contact information For research use only Not for use in diagnostic procedures P N WH112 Rev 1 2010 Affymetrix Inc All rights reserved Affymetrix Axiom Command Console DMET GeneAtlas GeneChip GeneChip compatible GeneTitan Genotyping Console myDesign NetAffx Powered by Affymetrix Procarta and QuantiGene are trademarks or registered trademarks of Affymetrix Inc Luminex and xMAP are registered trademarks of Luminex Corp All other trademarks are the property of their respective owners
11. or cultured cells Cells were plated in a 96 well plate at a density such that confluency was between 80 and 95 percent throughout the procedure Dependent on the cell type used wells were precoated to increase cell attachment to the surface Cultured cells were washed two times in 150 uL of 1x phosphate buffered saline PBS to remove traces of culture medium To fix cells 60 uL of 4 percent formaldehyde was added to each well and the plate was incubated for 30 minutes at room temperature The formaldehyde solution was removed by gently washing cells with 150 uL of 1x PBS three times To permeabilize cells 60 uL of working detergent solution was added to each well and the plate was incubated for 3 minutes at room temperature Wells were washed once in 150 uL of 1x PBS before adding 60 uL of protease solution After 10 minutes at room temperature cells were gently washed three times with 150 uL of 1x PBS To stop further permeabilization 60 uL of protease stop buffer was added to each well and the plate was incubated for up to 30 minutes All well contents were removed by expulsion before proceeding to probe set hybridization Sample preparation for FFPE tissue sections FFPE samples fixed in neutral buffer formalin for 16 24 hours at room temperature were sectioned at 4 to 6 um thickness and mounted onto positively charged Superfrost slides Fisher Scientific USA Tissue sections per slide were no larger than 22 mm x 22 mm Slides were incu
12. r A cultured cells and B FFPE tissue sections Figure 2A Target mRNA specific probe sets Z TYPE 4 blue Z TYPE 6 green Z TYPE 8 orange PreAmp 4 PreAmp 6 Amp 4 PreAmp 8 Amp6_ Label probe 4 Amp8 Label probe 6 Label probe 8 _ mRNA2 mRNA3 t Step 1 Prepare sample Step 2 Hybridize probe sets Step 3 Amplify signal Step 4 Image Step 1 Prepare sample Adherent cells on a solid surface are fixed and permeabilized Step 2 Hybridize probe sets Gene specific probe sets hybridize to target mRNAs For clarity a three plex assay with only single oligonucleotide pairs are shown however a typical probe set contains 20 or more oligonucleotide pairs Each probe set type e g type 4 interacts specifically with a corresponding signal amplification system e g PreAmp 4 Amp 4 Label Probe 4 to generate signal for visualization Step 3 Amplify signal Independent but compatible signal amplification systems enable simultaneous detection of multiple RNAs in a single assay Distinct sets of Preamplifier PreAmp Amplifier Amp and Label Probe LP molecules are used to detect different target mRNAs A PreAmp molecule hybridizes to each Probe Set oligonucleotide pair then multiple Amp molecules hybridize to each PreAmp Finally multiple LP oligonucleotides conjugated to fluorescent dyes hybridize to each Amp Step 4 Image Target mRNAs are visualized using a standard fluoresce
13. was decanted and the slides were washed by submersion and agitation in 1x PBS After three washes the slides were transferred into 4 percent formaldehyde for 5 minutes followed by rinsing once in 1x PBS Hybridization of probe sets for cultured cells Probe sets were diluted 1 100 to form a working probe set solution To hybridize the probe set 60 uL of diluted probe set was added to each well and the plate was incubated for 3 hours at 40 C Then wells were washed three times with 150 uL of 1x PBS buffer and the contents completely expelled For signal amplification 60 uL of prewarmed Preamplifier reagent PreAmp was added to the wells and the plate was incubated at 40 C for 60 minutes Wells were subsequently washed with 150 uL of 1x PBS three times before adding 60 uL of Amplifier reagent Amp The plate was incubated at 40 C for 60 minutes before the unbound reagent was removed by three washes with 150 uL of 1x PBS To visualize 60 uL of Label Probe reagent LP was added to each well and the plate was incubated at 40 C for 60 minutes Following three washes with 150 uL of 1x PBS nuclei were counterstained with DAPI 10 mg mL at a 1 10 000 ratio After 1 minute incubation at room temperature the DAPI was removed by washing once with 1x PBS To visualize 150 uL of fresh 1x PBS was added to each well and the plate was examined under a fluorescent microscope using the following filter sets FITC Ex 501 nm Em 523 nm Cy3 Ex 554 nm Em 576 nm
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