TissueScan Normal qPCR Arrays - cDNA panels (Human
Contents
1. Total RNA was isolated and subjected to oligo dT selection The recovered poly A RNA was then examined by Northern blot hybridization to confirm RNA integrity The poly A RNA was used to synthesize first strand cDNA using oligo dT primers and M MLV reverse transcriptase Individual cDNA pools were confirmed to be free of genomic DNA contamination and to contain complete reverse transcripts of selected rare and long mRNAs such as those for the transferrin receptor 5 kb and the ataxia telangiectasia gene 9 4 kb The first strand cDNAs from each tissue were then subjected to normalization such that they all contain an equivalent concentration of GAPDH cDNA for human and mouse samples or RpL32 cDNA for Drosophila samples Experimental Procedures Real time PCR Real time PCR continuously quantifies the PCR product throughout the entire thermocycling process Therefore it eliminates the need for agarose gel quantification and avoids the issue of saturated images of PCR products normally encountered in standard PCR quantitation This is achieved via comparison of the data during the linear amplification phase TaqMan probe 3 and SYBR Green 4 5 based protocols are the most commonly used real time PCR protocols These methods generate fluorescent signals that are proportional to the amount of the PCR products generated at the end of each cycle Differences among them involve the types of signals and the method by which the fluorescent signa2. C 2 Bone Marrow E 2 Pericardium C 3 Brain E 3 Pituitary C 4 Cervix E 4 Placenta C 5 Colon E 5 Prostate C 6 Descending part of duodenum E 6 Rectum C 7 Epididymis E 7 Retina C 8 Esophagus E 8 Seminal Vesicles C 9 Fat E 9 Skin C 10 Heart E 10 Spinal Cord C 11 Intestine Small E 11 Spleen C 12 Intracranial Artery E 12 Stomach D 1 Kidney F 1 Testis D 2 Liver F 2 Thymus D 3 Lung F 3 Thyroid D 4 Lymph Node F 4 Tongue D 5 PBL plasma blood leucocytes ER Tonsil D 6 Mammary gland F 6 Trachea D 7 Muscle F 7 Urethra D 8 Nasal Mucosa F 8 Urinary Bladder D 9 Optic Nerve F 9 Uterus D 10 Ovary F 10 Uvula D 11 Oviduct F 11 Vagina D 12 Pancreas F 12 Vena Cava Figure 2 HMRT102 Product Quality Control 10 0 10 1 A i H t IER Pie Figure 2 The Human Major Tissue qPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using GAPDH primers The GAPDH amplification products were detected using the SYBR Green protocol then run on an agarose gel The sample numbers in the bottom panel correspond to the cDNA templates listed in Figure 1 Top panel Real time PCR results fluorescence readings Lower panel PCR products on 2 agarose gel stained with ethidium bromide 11 Figure 3 HMRT102 Product Validation 800 0 700 0 600 0 500 0 Relative 400 0 Expression Level 300 0 200 0 100
3. pmol uL 26 uL 38 uL 50 uL 10 pmol well Reverse primer 10 pmol uL 26 uL 38 uL 50 uL 10 pmol well Tag DNA polymerase 5 U uL 2 6 uL 3 8 uL 5 uL 0 5 U well dd OO 569 4 uL 832 2 uL 1095 uL Total 780 uL 1140 uL 1500 uL e Remove the sealing film from the TissueScan plate Aliquot 30 uL of the PCR pre mix into each well avoiding cross contamination during pipetting This can best be achieved using a multichannel pipettor e Cover the top of the plate with a new adhesive cover sheet provided Seal each well tightly by pressing the cover around each well Remove any air bubbles that may have been trapped at the bottom of the tubes by gently tapping the plate on a flat surface Incubate the plate on ice for 15 minutes to allow the dried cDNA to dissolve Gently vortexing the plate facilitates resuspension of the DNA e Mount the plate snugly into the block of a 96 well thermal cycler so that each well makes tight contact with the heating unit Tighten the lid of the thermal cycler to ensure direct contact between the lid and the top of the plate this will reduce the chance of evaporation from the wells These steps are very critical for even template amplification e Program the thermal cycling conditions optimized for your gene specific primers e The following cycling conditions are listed here as a reference we strongly suggest that the cycling conditions be optimized for your gene specific primers Pre soak 94 C for 3 min 33 cycles of De
4. 0 0 0 TANO ee OANA EHNEN Sample number Figure 3 The Human Major Tissue qPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using primers designed against the human thyroglobulin gene The sample numbers in the bottom panel correspond to the cDNA templates listed in Figure 1 Top panel Real time PCR results fluorescence readings Lower panel Normalized relative expression levels among 48 tissue samples 12 Figure 4 TissueScan Human Brain Tissue qPCR Array HBRT101 CARER ER EE EEE SF e eseeeceaeeaece GOOOOOOOOOOC CX Se 123 456 78 9101112 Location Tissue Location Tissue C 1 Frontal Lobe D 1 Caudate C 2 Temporal Lobe D 2 Putamen C 3 Occipital Lobe D 3 Substantia Nigra C 4 Parietal Lobe D 4 Pituitary Gland C 5 Paracentral Gyrus D 5 Cerebellum grey C 6 Postcentral Gyrus D 6 Cerebellum white C 7 Olfactory Bulb D 7 Cerebellum vermis C 8 Thalamus D 8 Nucleus Accumbens C 9 Corpus Callosum D 9 Pons C 10 Hypothalamus D 10 Medulla C 11 Amygdala D 11 Spinal Cord C 12 Hippocampus D 12 Choroid Plexus Figure 5 HBRT101 Product Quality Control real time PCR Figure 5 The Human Brain Tissue gPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using GAPDH primers The GAPDH amplification products were detected using the SYBR Green protocol 14 Figure 6 HBRT101 Product Validation real time PCR 70
5. 0 Relative 600 Expression Level 400 200 Sample number Figure 15 The Drosophila Tissue qPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using primers designed against the doublesex gene The sample numbers in the bottom panel correspond to the cDNA templates listed in Figure 13 Top panel Real time PCR results fluorescence readings Lower panel Normalized relative expression levels among 12 tissue samples 24 Figure 16 TissueScan Rat Normal Tissue qPCR Array RNRT101 123 456 78 9101112 It o mm Go D gt Location Tissue Location Tissue C 1 Adipose Tissue E 1 Lymph Nodes C 2 Adrenal Gland E 2 Mammary Gland day1 of involution C 3 Aorta E 3 Mammary Gland day1 of lactation C 4 Bladder E 4 Mammary Gland non pregnant C 5 Brain Brainstem E 5 Mammary Gland pregnant E12 C 6 Brain Cerebellum E 6 Mammary Gland pregnant E16 C 7 Brain Cerebral Cortex E 7 Ovary C 8 Brain Front Cortex E 8 Pancreas C 9 Brain Medulla E 9 Pituitary C 10 Brain Midbrain E 10 Placenta C 11 Brain Pons E 11 Prostate C 12 Brain Posterior Cortex E 12 Skeletal Muscle D 1 Brain Thalamus F 1 Skin D 2 Brain Whole F 2 Spinal Cord D 3 Embryo E12 F 3 Spleen D 4 Embryo E15 F 4 Stomach D 5 Embryo E17 F 5 Striatum D 6 Embryo E19 F 6 Testis D 7 Epididymis F 7 Thymus D 8 Esophagus F 8 Thyroid D 9 Heart F 9 Trachea D 10 Intestin
6. 0 00 600 00 500 00 Relative 400 00 Expression Level 300 00 200 00 100 00 0 00 e DN Oo st UO OR ODO e Sample number Figure 6 The Human Brain Tissue qPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using primers designed against the human growth hormone releasing hormone receptor GHRHR NM_001009824 The sample numbers in the bottom panel correspond to the cDNA templates listed in Figure 4 Top panel Real time PCR results fluorescence readings Lower panel Normalized relative expression levels among 48 tissue samples 15 Figure 7 HBRT101 Product Quality Control and Validation standard PCR 12 3 4 5 6 7 8 9 10 11 12 13 14 15 1617 18 19 20 21 22 23 24 OO Go Figure 7 The Human Brain Tissue qPCR Array used in a standard PCR protocol using the primers indicated below The amplification products were run on an agarose gel stained with ethidium bromide The sample numbers in each panel correspond to the cDNA templates listed in Figure 4 Row 1 Row 2 Row 3 Row 4 Row 5 Row 6 NM_001101 Homo sapiens GAPDH NM_001009824 Homo sapiens growth hormone releasing hormone receptor GHRHR NM_033207 Homo sapiens transmembrane protein 10 NM_000816 Homo sapiens gamma aminobutyric acid GABA A receptor NM_005458 Homo sapiens GABA B receptor NM_000832 Homo sapiens NMDA receptor subunit NR1 Figure 8 TissueScan Mouse Developmental Tissue qPCR Array MDRT101 123 456 78 91011
7. 12 rt o mm Oo D gt Location Tissue Location Tissue Embryo Day 13 Postnatal Day 7 C 1 Telencepahlon Diencephalon D 11 Frontal Cortex C 2 Mesencephelon Midbrain D 12 Posterior Cortex C 3 Rhombencephalon Hindbrain E 1 Entorhinal Cortex C 4 Spinal Cord E 2 Olfactory Bulb Embryo Day 15 E 3 Hippocampus C 5 Telencephalon E 4 Striatum C 6 Diencephalon E 5 Thalamus C 7 Midbrain E 6 Hypothalamus C 8 Pons E 7 Cerebellum C 9 Medulla E 8 Midbrain C 10 Spinal Cord E 9 Pons Embryo Day 18 E 10 Medulla C 11 Frontal Cortex E 11 Spinal Cord C 12 Posterior Cortex Adult 5 Week D 1 Entorhinal Cortex E 12 Frontal Cortex D 2 Olfactory Bulb F 1 Posterior Cortex D 3 Hippocampus F 2 Entorhinal Cortex D 4 Striatum F 3 Olfactory Bulb D 5 Thalamus F 4 Hippocampus D 6 Hypothalamus F 5 Striatum D 7 Midbrain F 6 Thalamus D 8 Pons F 7 Hypothalamus D 9 Medulla F 8 Cerebellum D 10 Spinal Cord F 9 Midbrain F 10 Pons F 11 Medulla F 12 Spinal Cord Figure 9 MDRT101 Quality Control real time PCR 10 14 10 0 e Gg 10 2 O 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle Figure 9 The TissueScan Mouse Developmental Tissue qPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using GAPDH primers The GAPDH amplification products were detected using the SYBR Green protocol then run on an agaros
8. Table of Contents Table of Contents sce sto cttin es E E 1 Package Contents and Storage Conditions 2 INTFOQUCTION Ait tiedec tice aan Seether nthe onthe vice tea diene than ee the tee thee 3 Production and Quality Aesurance cee eeeeaeeeeaeeeeeeeeceaeeeeaaeseeeeesaaeeesaaesdeaeeseaeeesaeeetaeeeenees 4 Experimental Procedures AAA 4 Real time PGR Ree eer dE EELER SEET gd ailen faecal 4 Real time POR ProCe Cure ee ee DEE EEN Eeer dEr EE ed 5 StandardiPGR WEE 6 Troubleshooting Guld EE 8 References issida ate elie dead A canara Eed a BA ae 8 PIQUICS otters erin Ata Sct tha te Re yates cite atts A SE EAE cae eee en bad 8 iu koe 10 Figure 1 TissueScan Human Major Tissue qPCR Array HMPDTIO 10 Figure 2 HMRT102 Product Quality Control 11 Figure 3 AMR 102 Product Valisen deck delt nna atin nine cred ancien helen 12 Figure 4 TissueScan Human Brain Tissue qPCR Array HBRT101 0 eeeeeeeeeeeneeeeeeeeeneeeeeeeeeaeeeeeeeeaeeeeeeeeenes 13 Figure 5 HBRT101 Product Quality Control real time DCH 14 Figure 6 HBRT101 Product Validation real time DCH 15 Figure 7 HBRT101 Product Quality Control and Validation Standard DCH 16 Figure 8 TissueScan Mouse Developmental Tissue qPCR Array MDPTIO 17 Figure 9 MDRT101 Quality Control real time DCH 18 Figure 10 MDRT101 Product Quality Control and Validation standard DCH 19 Figure 11 TissueScan Mouse Normal Tissue qPCR Array MNRT101 uu eeecceeeeeeeeeeeneeeeeeeeeee
9. al cycler so that each well makes tight contact with the heating unit Tighten the lid of the thermal cycler to ensure direct contact between the lid and the top of the plate this will reduce the chance of evaporation from the wells These steps are very critical for even template amplification e Program the thermal cycling conditions optimized for your gene specific primers e Set the reporter type TaqMan probe or SYBR Green and reference dye according to the equipment user s guide If a SYBR Green protocol is used program a post amplification melting curve test if possible e The following cycling conditions are listed here as a reference we strongly suggest that the cycling conditions be optimized for your gene specific primers Activation 50 C for 2 min Pre soak 95 C for 5 min 42 cycles of Denaturation 95 C for 15 sec Annealing 60 C for 1 min Note for an amplicon gt 120 bp a 72 C extension step must be incorporated e Analyze the expression data using the software provided by the thermal cycler manufacturer In most cases relative expression levels can be calculated 6 Use your own preferred method to perform this analysis e Examine the PCR products via 2 agarose gel if desired recommended when a SYBR Green protocol is used for detection Standard PCR Although the TissueScan Tissue qPCR Array is designed for real time PCR procedures it can also be used in a standard PCR protocol using agarose gel elec
10. com support citations gene_expression mspx With the adoption of real time PCR technology many researchers expressed a need for a real time PCR or quantitative PCR qPCR version of the RapidScan product To accommodate this need we have developed the TissueScan Tissue qPCR Arrays using the same high quality cDNAs as those used in the RapidScan panels OriGene s TissueScan Real Time qPCR Arrays have gone through vigorous quality control and validation Unlike the RapidScan gene expression panels the TissueScan qPCR arrays contain only one concentration of cDNAs normalized to a housekeeping gene Benefits of the Tissue qPCR Array include e Fast and non radioactive no radiolabeling of probe no blot hybridization no filter washing e Instant expression data no agarose gel electrophoresis required e Simultaneous examination of a wide range of different tissues and or developmental stages e Highly sensitive and quantitative e Extremely reliable Production and Quality Assurance TissueScan Tissue qPCR Arrays were assembled by selecting frequently studied tissues and or developmental stages To avoid detection of individual differences in gene expression tissues were pooled whenever possible from multiple individuals For the human panels tissues were selected from individuals of different ethnicity For the mouse panels the tissues were derived from NIH Swiss mice The Drosophila panels were derived from samples from the Canton S strain
11. d non specific background noise The non specific components may be reduced by increasing the stringency of the PCR amplification References 1 Khong HT Rosenberg SA The Waardenburg syndrome type 4 gene SOX10 is a novel tumor associated antigen identified in a patient with a dramatic response to immunotherapy Cancer Res 2002 Jun 1 62 11 3020 3 2 Egland KA Kumar V Duray P Pastan Characterization of overlapping XAGE 1 transcripts encoding a cancer testis antigen expressed in lung breast and other types of cancers Mol Cancer Ther 2002 May 1 7 441 50 Lee LG Connell CR Bloch W 1993 Allelic discrimination by nick translation PCR with fluorogenic probes Nucleic Acids Res 11 21 16 3761 6 T B Morrison J J Weis C T 1998 Quantification of Low Copy Transcripts by Continuous SYBR Green Monitoring during Amplification BioTechniques 24 954 1998 K M Ririe R P Rasmussen C T Wittwer 1997 Product Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction Anal Biochem 245 154 M W Pfaffl A new mathematical model for relative quantification in real time RT PCR 2001 Nucleic Acids Research 2001 29 2002 2007 Figures Figure 1 TissueScan Human Major Tissue qPCR Array HMRT102 rt o mm Oo D gt 123 456 78 9101112 Location Tissue Location Tissue C 1 Adrenal Gland E 1 Penis
12. e F 10 Uterus non pregnant D 11 Kidney D 12 Liver 25 Figure 17 RNRT101 Product Quality Control 10 1 limes 10 3 O 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle Figure 17 The Rat Normal Tissue qPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using GAPDH primers The GAPDH amplification products were detected using the SYBR Green protocol then run on an agarose gel The sample numbers in the bottom panel correspond to the cDNA templates listed in Figure 16 Top panel Real time PCR results fluorescence readings Lower panel PCR products on 2 agarose gel stained with ethidium bromide 26 Figure 18 RNRT101 Product Validation Relative Expression Level 700 600 500 400 300 200 100 0 Sample number Figure 18 The Rat Normal Tissue qPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using primers designed against the rat LOC299907 XM_216920 gene The sample numbers in the bottom panel correspond to the cDNA templates listed in Figure 16 Top panel Real time PCR results fluorescence readings Lower panel Normalized relative expression levels among 46 tissue samples 27
13. e gel Top panel Real time PCR results fluorescence readings Lower panel PCR products on 2 agarose gel stained with ethidium bromide Figure 10 MDRT101 Product Quality Control and Validation standard PCR a ee ee 2 E S Figure 10 The TissueScan Mouse Developmental Tissue qPCR Array used in a standard PCR protocol using the primers indicated below The amplification products were run on an agarose gel stained with ethidium bromide Row 1 NM_001101 Mouse GAPDH Row 2 NM_003235 Mouse Hox 3 1 Row 3 NM_014357 Mouse Dopamine receptor D3 19 Figure 11 TissueScan Mouse Normal Tissue qPCR Array MNRT101 123 456 78 9101112 KA A RS SS KM Zomm oo mp gt Location Teens Location Teens Cl JL Adipose E1 f Placenta C 2 Adrenal Gland 1 E2 Prostate C3 Gadder ES Skin C 4 Bone Marrow E4 Smal Intestine Ch Brain E5 f Spinal Cord CS Coon CES f Spleen C7 Embryo dy IT Ei Stomach CH Embryo df E8 Testis CH Embryo d E9 Thymus C 10 Embryodi7 E10 Thyroid CL Epididymus E11 Trachea C 12_ Esphogueal E12 Uterus DI Eye D 2 D 3 D 4 D 5 D 6 D 7 D D 9 D 10 Di Muscle D 12 20 Figure 12 MNRT101 Quality Control Figure 12 TissueScan Mouse Normal Tissue used in a real time PCR protocol in a PE7700 thermal cycler using GAPDH primers The GAPDH amplification products in were detected using the SYBR Green p
14. eeeeeeeeeeeeeeeeaees 20 Figure 12 MNRT101 Quality Control 21 Figure 13 TissueScan Drosophila Tissue qPCR Array DSRT101 ee eeecceeeeeeeeneeeeeeseeeeeeeeeeeeeseeeeeeeeeeeeenees 22 Figure 14 DSRT101 Product Quality Control 23 Figure 15 DSRT101 Prod ct Validation iss cecden s edceesvaevecet fh Rents cones ete eevee he dats cee ie ane th Bearers 24 Figure 16 TissueScan Rat Normal Tissue QPCR Array DNDTIOTI 25 Figure 17 RNRT101 Product Quality Control 26 Figure 18 RNRT101 Product Validation AA 27 Package Contents and Storage Conditions The following components are included e Identical sealed TissueScan Tissue qPCR Array plates containing dried PCR ready first strand cDNAs e Adhesive cover sheets for sealing the plates e Control primers TissueScan Cat Unique of plates Control primers Tissue qPCR samples provided Arrays format Human Major Tissue HMRT102 48 4x12 2 GAPDH Human Major Tissue HMRT502 48 4x12 10 GAPDH Human Brain HBRT101 24 2x12 2 GAPDH Human Brain HBRT501 24 2x12 10 GAPDH Mouse Developmental MDRT101 48 4x12 2 GAPDH Mouse Developmental MDRT501 48 4x12 10 GAPDH Mouse Normal MNRT101 36 3X12 2 GAPDH Mouse Normal MNRT501 36 3X12 10 GAPDH Rat Normal RNRT101 46 4X12 2 GAPDH Rat Normal RNRT501 46 4X12 10 GAPDH Drosophila DSRT101 12 2 RpL32 Drosophila DSRT501 12 10 RpL32 The above components are shipped at room t
15. emperature but should be kept at 20 C for long term storage If properly stored they have a 12 month shelf life The following components are required but are not provided Reagents for real time PCR amplification e g 2x master mix Gene specific primers Gene specific probe if using a TaqMan Assay A 96 well real time thermocycler The polymerase chain reaction PCR is protected by patents held by Hoffmann La Roche Purchase of any of OriGene s PCR related products does not convey a license to use the PCR process covered by these patents Purchasers of these products must obtain a license before performing PCR NOTE FOR RESEARCH PURPOSES ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USAGE Introduction Gene expression profiling is one of the most frequently used approaches for characterizing novel transcripts either derived from expressed sequence tag EST projects or the completed human and mouse genomes by means of program prediction The amount of transcript accumulated in a particular tissue normal or diseased often provides valuable information about the function of the gene Many technologies have been developed for obtaining gene expression data including Northern blotting RT PCR and DNA microarray analysis Each of the technologies has its advantages and disadvantages The Northern blot method is the only method that reveals the size information of a transcript as well as transcript abundance The drawback of the method
16. is that it is low throughput and frequently involves radioactive agents DNA microarray analysis is a well established gene expression profiling technology The primary advantage of DNA microarrays is that it is able to simultaneously generate the expression profiles of thousands of genes Although DNA microarrays have produced a large amount of useful gene expression data their sensitivity and accuracy are sometimes compromised by non specific hybridizations due to shared homology among different genes Many researchers use RT PCR to validate the data from microarray experiments RT PCR long used for gene expression profiling is fast and sensitive Instead of using hybridization RNA transcripts are converted to the complementary DNA cDNA using a reverse transcription reaction and the cDNAs are subsequently used as the templates for PCR amplification Thus the amount of PCR product will be correlated to the amount of the original RNA transcript Previously OriGene Technologies Inc developed a unique PCR based system the RapidScan Gene Expression Panel for fast and accurate gene expression profiling With serially diluted cDNA templates the relative amounts of transcript in different tissues could be easily obtained by standard PCR experiments and subsequent agarose gel electrophoresis This system has been well adopted by the research community Examples of successful panel usage are cited in numerous publications 1 2 amp http www origene
17. ls are generated In a TaqMan protocol a pair of gene specific primers and a sequence specific fluorogenic probe with reporter and quencher is included in the PCR mix The specific probe anneals proportionally to the single stranded DNA and as DNA synthesis occurs is subsequently removed base by base by the 5 exonuclease activity of Taq polymerase Consequentially the released fluorescent reporter is freed from the quencher and generates a fluorescent signal that is proportional to the amount of PCR product being accumulated This method is very sensitive and reliable A SYBR Green protocol on the other hand does not require a fluorescent probe SYBR Green binds to double stranded DNA to generate detectable fluorescence and the amount of signal is proportional to the amount of double stranded DNA in solution Since SYBR Green binds indiscriminately to double stranded DNAs it will generate false signals if non specific elements e g primer dimers exist This is frequently used in many laboratories due to its simplicity and low cost Both TaqMan and SYBR Green protocols can be used with the TissueScan Tissue qPCR Array Gene specific primers and probes A successful real time PCR detection requires a set of sequence specific primers and may require a fluorescent probe as described above Many primer and probe designing programs are commercially available or are found as freeware on the internet It is suggested that qPCR amplicons
18. naturation 94 C for 30 sec Annealing 55 C for 30 sec Extension 72 C for 2 min Soak 72 C for 5 min Use 31 cycles for the control primers and run a 2 agarose gel to obtain the results shown in Fig 3 For your target gene set the cycle number based on the abundance of the transcript if known This is the suggested annealing temperature for the GAPDH control primers The annealing temperature should be 5 C below that of the Tm of the gene specific primers e When the reaction is completed remove the cover sheet and add to each well 15 uL of 3X DNA loading buffer Mix gently by pipetting up and down e Load 15 uL of each sample onto the gel Store the remaining samples at 20 C e Separate the samples by electrophoresis and document the results Positive control PCR Two identical TissueScan plates are provided for your convenience Ideally the two plates may be used for the analysis of two different genes or ESTs Alternatively after obtaining an adequate expression profile for a test gene the second plate may be used for determining the expression profile using the control primers provided To perform the control PCR substitute the gene specific primers with 100 uL of the control primers GAPDH for human and mouse panels RpL32 for Drosophila panels The single primer tube contains both the forward and reverse primers These primer pair has been optimized for a SYBR Green protocol The expected sizes of the PCR products f
19. ols Stock solution 2X master mix Forward primer 10 pmol uL Reverse primer 10 pmol uL ddH2O Total For TaqMan protocols Stock solution 2X master mix Forward primer 10 pmol uL Reverse primer 10 pmol uL 20X TaqMan probe ddH2O Total 24 samples Volume 390 uL 26 uL 26 uL 338 uL 780 uL 24 samples Volume 390 uL 26 uL 26 uL 39 uL 299 uL 780 uL 36 samples Volume 570 uL 38 uL 38 uL 494 uL 1140 uL 36 samples Volume 570 uL 38 uL 38 uL 57 uL 437 uL 1140 uL 48 samples Volume 750 uL 50 uL 50 uL 650 uL 1500 uL 48 samples Volume 750 uL 50 uL 50 uL 75 uL 575 uL 1500 uL Final 1X 10 pmol well 10 pmol well Final 1X 10 pmol well 10 pmol well 1X e Remove the sealing film from the plate Aliquot 30 uL of the PCR pre mix into each well avoiding cross contamination during pipetting This can best be achieved using a multi channel pipettor after equally dividing the 1 5 ml premix into PCR tubes e Cover the top of the plate with a new adhesive cover sheet provided Seal each well tightly by pressing the cover around each well Remove any air bubbles that may have been trapped at the bottom of the tubes by gently tapping the plate on a flat surface Incubate the plate on ice for 15 minutes to allow the dried cDNA to dissolve Gently vortexing the plate will facilitate resuspension of the DNA e Mount the plate snugly into the 96 well real time therm
20. rom the control primers are listed below human GAPDH product 151 bp mouse GAPDH product 151 bp Drosophila RpL32 product 434 bp Sample profiling The cDNAs in every TissueScan qPCR panel have been vigorously quality controlled The cDNAs were normalized to GAPDH or RpL32 content and tested to confirm normalization using those same primers with a SYBR Green protocol Figs 2 5 9 12 14 The panels are also tested both in real time PCR and in standard PCR Figs 3 6 7 10 15 Troubleshooting Guide No PCR product detected May have omitted a PCR component Be sure to use a written checklist when assembling the reaction mix May have used an inappropriate annealing temperature If at all possible test primers against a known template e g diluted cloned DNA to determine the optimal amplification conditions May be due to poor primer design Follow the general guidelines when designing PCR primers More than one PCR product detected on an agarose gel It is possible that all of the products are specific resulting from the amplification of either alternatively spliced mRNAs or transcripts derived from distinct members of a gene family This may be resolved by either DNA sequencing of the PCR products or by using nested PCR primers for reamplification Detection of both specific band s and a background smear This may be the result of the annealing temperature used in the experiment giving rise to both specific component s an
21. rotocol 21 Figure 13 TissueScan Drosophila Tissue qPCR Array DSRT101 123 456 78 9101112 e e e e e e e e e e e Zomm oo mp gt C 1 Embryo 0 4 hours D 1 Embryo 0 4 hours C 2 Embryo 4 8 hours D 2 Embryo 4 8 hours C 3 Embryo 8 12 hours D 3 Embryo 8 12 hours C 4 Embryo 12 24 hours D 4 Embryo 12 24 hours C 5 ist instar D 5 1st instar C 6_ 2nd instar D 6 2nd instar C 7 3rd instar D 7 3rd instar C 8 Pupae D 8 Pupae C 9 Male eye head D 9 Male eye head C 10 Female eye head D 10 Female eye head C 11 Male body D 11 Male body C 12 Female body D 12 Female body 22 Figure 14 DSRT101 Product Quality Control D 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle Ee ee ee wm ea Figure 14 The Drosophila Tissue qPCR Array used in a real time PCR protocol in a PE7700 thermal cycler using primers designed against ribosomal protein RpL32 The amplification products were detected using the SYBR Green protocol then run on an agarose gel Top panel Real time PCR results fluorescence readings Lower panel PCR products on 2 agarose gel stained with ethidium bromide 23 Figure 15 DSRT101 Product Validation 1200 1000 80
22. should range from 80 150 base pairs in length When using the SYBR Green protocol since the fluorescence signal reflects the total amount of double stranded DNA including specific and non specific products it is critical to design primers that maximize specific products and minimize non specific products We recommend optimizing PCR primers and conditions using known templates before the PCR system is used with the TissueScan Tissue qPCR Array PCR master mix Both TaqMan and SYBR Green master mixes are commercially available and are recommended for use in the TissueScan Tissue qPCR Array A real time PCR master mix usually contains PCR buffer magnesium dNTPs and Taq polymerase Most commercially available real time master mixes are provided as a 2X concentration with a reference dye ROX included Real time PCR thermocycler A 96 well real time thermocycler and detection system is required for using the TissueScan Tissue qPCR Array The machines listed in the following table are compatible with these panels For devices not listed in the table please contact OriGene for suggestions Manufacturer Model ABI Prism 7000 7700 7300 7500 9500 Bio Rad iCycler MyiQ Stratagene Mx4000 Mx3000p Mx3005p Real time PCR procedure Remove the TissueScan plate from 20 C storage and allow it to warm to room temperature Prepare a pre mix in a 1 5 ml tube according to the recipe below For SYBR Green protoc
23. trophoresis Semi quantitative analysis is desirable when researchers prefer to use gel documentation data for publication An experimental procedure for standard PCR is described below Primer design for standard PCR Primer design is a critical factor in obtaining success in PCR There are no simple rules however the general guidelines for primer design indicate that primers should be between 17 and 25 nucleotides in length have a G C content of approximately 50 and should not form strong secondary structures Avoid sequences that are susceptible to primer dimer formation In addition the primers should ideally correspond to the coding region of the gene of interest The positions of the forward and the reverse primers should be between 300 and 1000 bp apart so as to facilitate efficient template amplification and easy detection of the PCR product in an agarose gel If possible a pilot PCR amplification should be performed using the gene specific primers and a test template such as a plasmid cDNA clone or a first strand cDNA preparation that is known to contain the target sequence of interest Standard PCR procedure Remove the TissueScan plate from 20 C storage and allow it to warm to room temperature Prepare a PCR pre mix according to the following recipe 24 samples 36 samples 48 samples Stock solution Volume Volume Volume Final 10X PCR buffer 78 uL 114 uL 150 uL 1X dNTPs 2 mM each 78 uL 114 uL 150 uL 0 2 mM Forward primer 10
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