EZ-GENXpress TM Normalized Gene Expression Detection Kit (Cat. #: EP-10001, EP & EP-10003)

Size: px
Start display at page:

Download "EZ-GENXpress TM Normalized Gene Expression Detection Kit (Cat. #: EP-10001, EP & EP-10003)"

Transcription

1 EZ-GENXpress TM Normalized Gene Expression Detection Kit (Cat. #: EP-10001, EP & EP-10003) INSTRUCTION MANUAL *These products are designed and sold for use in the Polymerase Chain Reaction (PCR) process covered by patents owned by Hoffman-LaRoche. Use of the PCR process requires a license. A license for diagnostic purposes may be obtained from Roche Molecular System. A license for research may be obtained by the purchase and the use of authorized reagents and DNA thermocyclers from the Perkin-Elmer Corporation or by otherwise negotiating a license with Perkin-Elmer. This product is intended for research use only and not for diagnostic purposes. M B I Maxim Biotech, Inc. 780 Dubuque Avenue Tel: (800) So. San Francisco, CA Fax: (650) U.S.A. Website: October, 2000 GEN

2 I. Introduction Molecular characterization of any gene usually includes a thorough analysis of the temporal and spatial distribution of RNA expression. Northern blot and RNase Protection Assay are the widely used procedures for determining the abundance of a particular mrna in a total or poly(a) sample. The RT-MPCR provides an alternative and accurate way to detect gene expression as it amplifies all genes in same condition. The variations in RNA isolation, initial quantitation errors, and tube-to-tube variation in RT and PCR can be compensated by co-amplification of specific gene with one of house-keeping genes such as GAPDH (multiplex PCR, MPCR). The difference in gene expression can be determined by normalizing its expression against GAPDH expression. Maxim's Normalized Gene Expression Detection Kit provides reagents to make conventional RT-PCR quantitative and is designed for optimization of co-amplification of your specific genes with a well-designed GAPDH primers. The kit includes Maxim's properitary MPCR Optimizer system so that most of genes can be co-amplified with GAPDH with minimal optimization. II. Key Factors Governing Gene Expression Detection By RT-MPCR (1) PCR Primers The key to successful PCR of any type lies in the design of appropriate primers. Ideally, all the primers in a multiplex reaction should enable identical amplification efficiencies for each amplicon. While it is difficult to predict the efficiency that any given primer pair will display, oligonucleotides with nearly identical optimum annealing temperatures should work under fairly similar condition. The Tm for GAPDH primers provided in Kit is 55 C. Because GAPDH is house-keeping gene and has high gene copy number, it is wise to design your primers with Tm 5 C-10 C higher than GAPDH primer's Tm, preferably 65 C-70 C and to use less GAPDH primers in MPCR so that PCR amplification of GAPDH is discrminated and your specific gene can be co-amplified. Other factor to consider when planning MPCR is to avoid 3'-end overlap among all primers because 3'-end overlaps usually favor the formation of "primer dimers". Multiple sets of primers increase the possibility of primer complementarity at the 3'-end, leading to "primer-dimers". Hot start PCR can decrease the formation of "primer-dimers" and is highly recommended. The primer concentration we recommended for MPCR is between 0.05 µm-0.1 µm. Concentration for GAPDH primers should be adjusted to be around one quarter of customer's specific primer concentration, usually 0.02 µm-0.03 µm. Most of PCR primers shall work well under this system as long as customer's primers don't generate same size of PCR products as GAPDH primers from Kit do. For high GC PCR, primers with higher Tm are recommended for use, preferably 75 C-80 C. The strand separation temperature for GC-rich DNA is significantly higher than normal DNA. The two strands of PCR amplicon intend to re-anneal faster at relatively lower temperature and compete with primer priming. The high re-anneal temperature in PCR will favor primer priming and therefore increase amplification efficiency. (2) Cycle Parameters The most important cycle parameters for Multiplex PCR are the annealing temperature and cycle numbers. The annealing temperature should be kept as high as possible and the fewest number of cycles as will permit easy detection of the products should be performed. The optimal annealing temperature should be obtained by optimizing each individual primer pair first. The lowest optimal annealing temperature among all primer pairs should be used in the Multiplex PCR. In contrast, the highest optimal annealing temperature should be used in high GC PCR. The higher denaturation temperature may also be used for high GC PCR. We recommend to use 95 C-96 C denaturation for first few cycles at least. (3) dntps Concentration Generally, high dntps concentrations should be used in Multiplex PCR. The 0.25 mm dntps are recommended for most of MPCR, although they may vary in each individual case. 2

3 (4) Enzymes High concentration of DNA Polymerase is required in MPCR. Taq DNA Polymerase and its derivatives work well under Maxim's MPCR Optimizer System. A concentration of 2.5 U Taq DNA Polymerase per 50 µl PCR reaction is recommended for MPCR, although different enzyme concentration may be required for each individual case. A mixture of Taq DNA Polymerase with Pfu DNA Polymerase from Stratagene at unit ratio of 15:1sometimes makes MPCR optimization easier, therefore is recommended when users encounter difficulty or intend to amplify longer DNA fragments. High concentration of DNA Polymerase is recommended for high GC PCR. Because 95 C-96 C denaturation steps are used for first few cycles, Taq DNA Polymerase may lost lots of its activities during later cycles. (5) MPCR Buffers 10 x MPCR Buffers all contain 250 mm Tris-HCl, ph 8.3, 500 mm KCl, various concentrations of Mg ++ and Taq DNA Polymerase Additive. Therefore, Taq DNA Polymerase and its derivatives all should work well under MBI's MPCR Buffer System. The Taq DNA Polymerase Additive is used to minimize the competition among amplicons during MPCR, therefore enhance MPCR performance. For high GC PCR, different amounts of DMSO and GC-Normalizer TM are added into MPCR Buffers to decrease temperature of DNA strand separation. (6) GC Content Some genes contain high GC that may prevent their amplification by standard PCR techniques. The strand separation temperature for high GC DNA is usually higher. The denaturation of DNA become difficult especially during later cycle when amplicom concentration become higher. Some reagents have been shown to facilitate DNA strand separation either because they disrupt base pairing (e.g. DMSO, formamide) or isostabilize DNA (e.g. TMAC). Some of these reagents have been included in PCR reactions to amplify GC-rich or secondary structure-rich templates 2,3. Normalized Gene Expression Detection Kit from Maxim incorporates DMSO and GC-Normalizer TM into Maxim's MPCR Optimizer Buffer System to help DNA strand separation of GC-rich templates. The special reagent GC-Normalizer TM in Kit is used to facilitate DNA strand separation with or without DMSO. The high GC PCR/MPCR can all be optimized in a single system. As a rule of thumb, We recommend to use 5% DMSO+1M GC-Normalizer TM if test samples contain 60-75% GC and 5% DMSO+2M GC-Normalizer TM if test samples contain 80-90% GC. III. Optimize your Multiplex PCR Set up 6 MPCRs with your test samples, primers, and GAPDH primers & MPCR Buffers 1-6 from Kit. If high GC-template, add proper amount of DMSO & GC-Normalizer TM into PCR mixture. 3

4 IV. Product: EZ-GENXpress TM Normalized Gene Expression Detection Kit (Human) Description The Normalized Gene Expression Detection Kit is designed for optimization of co-amplification of any specific human genes with a specially-designed GAPDH primers. The GAPDH primers have a Tm of 55 o C and can generate a 921 bp PCR products using human cdna. The 6 MPCR Buffers provided in the Kit ensure that the MPCR be optimized in hours. The GC- Normalizer TM is used to facilitate DNA strand separation of high GC template. Virtually any human genes can be co-amplified with GAPDH gene under this system as long as they have proper Tm, no serious 3'-end overlap, and don't generate same size of products as GAPDH primers do. Cat. No. EP-10001(GEN-1001) Rxns. 20 Storage Conditions: -20 o C Cat. No. Component Quantity MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl GCG-0001 GC-Normalizer TM 1.2 ml GCG-0002 DMSO 0.4 ml GAP-1001/3 GAPDH Primers (10X) 100 µl NTP-0001 dntps (2.5 mm each) 80 µl Sterile ddh 2 O 1.0 ml Instruction Manual 1 copy MPCR Optimization (1) Taq DNA polymerase and its derivative from Perkin Elmer is highly recommended for MPCR. NOTE: SPIN ALL TUBES BEFORE USE!! (2) Reaction Mixture Preparation I: Set up six PCR reactions with the test sample and MPCR Buffer 1 to Buffer 6 (Pink-Capped). If one or more the six buffers provide satisfactory results, there is no need to proceed further. II: If amplify high GC sequences, add proper amounts of DMSO and GC-Normalizer TM into PCR mixture. III: EDTA concentration in test sample must not exceed 0.5 mm because Mg ++ concentration in MPCR Buffers is limited to certain ranges, or add more Mg ++ into PCR mixture to compensate for EDTA. We strongly recommend to run a MPCR reaction with the positive control provided in the Kit. Since the MPCR DNA polymerase needed in each reaction is in a very small volume, it is recommended that all of the PCR components be premixed in a sufficient quantity for daily needs and then dispensed into individual reaction vials to achieve more accurate measurement. Hot start PCR is highly recommended. Per assay Add in order Distilled water 25.5 µl 10X MPCR Buffer 5.0 µl GAPDH Primers (10X) 5.0 µl Specifc Gene Primers (10X) 5.0 µl Taq DNA Polymerase (5 U/ µl) 0.5 µl cdna ( µg/µl) 5.0 µl dntp mixture (2.5 mm each) 4.0 µl DMSO GC-Normalizer TM Mineral oil 50.0 µl (Optional) (3) PCR thermocycle profile: Reaction profiles will need to be optimized according to the machine type and needs of user. A time-temperature profile for the positive control PCR optimized for Perkin Elmer machine type 480 or 9600 is provided as follows: Temperature Time Cycles 94 C 3' 1X 94 C 30" C 30" 30-35X 70 C 2' 70 C 10' 1X 25 C Soak (4) PCR Product Detection Mix 10 µl PCR product with 2 µl 6X loading buffer. Apply each to an agarose gel. Electrophoresis and photograph. Analyze the results and determine the optimal MPCR condition for a specific MPCR application. Each individual MPCR Buffer can also be purchased from MBI. 4

5 V. Product: EZ-GENXpress TM Normalized Gene Expression Detection Kit (Mouse) Description The Normalized Gene Expression Detection Kit is designed for optimization of co-amplification of any specific mouse genes with a specially-designed GAPDH primers. The GAPDH primers have a Tm of 55 o C and can generate a 532 bp PCR products using mouse cdna. The 6 MPCR Buffers provided in the Kit ensure that the MPCR be optimized in hours. The GC- Normalizer TM is used to facilitate DNA strand separation of high GC template. Virtually any human genes can be co-amplified with GAPDH gene under this system as long as they have proper Tm, no serious 3'-end overlap, and don't generate same size of products as GAPDH primers do. Cat. No. EP (GEN-2001) Rxns. 20 Storage Conditions: -20 o C Cat. No. Component Quantity MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl GCG-0001 GC-Normalizer TM 1.2 ml GCG-0002 DMSO 0.4 ml GAP-1101/2 GAPDH Primers (10X) 100 µl NTP-0001 dntps (2.5 mm each) 80 µl Sterile ddh 2 O 2.0 ml Instruction Manual 1 copy MPCR Optimization (1) Taq DNA polymerase and its derivative from Perkin Elmer is highly recommended for MPCR. NOTE: SPIN ALL TUBES BEFORE USE!! (2) Reaction Mixture Preparation I: Set up six PCR reactions with the test sample and MPCR Buffer 1 to Buffer 6 (Pink-Capped). If one or more the six buffers provide satisfactory results, there is no need to proceed further. II: If amplify high GC sequences, add proper amounts of DMSO and GC-Normalizer TM into PCR mixture. III: EDTA concentration in test sample must not exceed 0.5 mm because Mg ++ concentration in MPCR Buffers is limited to certain ranges, or add more Mg ++ into PCR mixture to compensate for EDTA. We strongly recommend to run a MPCR reaction with the positive control provided in the Kit. Since the MPCR DNA polymerase needed in each reaction is in a very small volume, it is recommended that all of the PCR components be premixed in a sufficient quantity for daily needs and then dispensed into individual reaction vials to achieve more accurate measurement. Hot start PCR is highly recommended. Per assay Add in order Distilled water 25.5 µl 10X MPCR Buffer 5.0 µl GAPDH Primers (10X) 5.0 µl Specifc Gene Primers (10X) 5.0 µl Taq DNA Polymerase (5 U/ µl) 0.5 µl cdna ( µg/µl) 5.0 µl dntp mixture (2.5 mm each) 4.0 µl DMSO GC-Normalizer TM Mineral oil 50.0 µl (Optional) (3) PCR thermocycle profile: Reaction profiles will need to be optimized according to the machine type and needs of user. A time-temperature profile for the positive control PCR optimized for Perkin Elmer machine type 480 or 9600 is provided as follows: Temperature Time Cycles 94 C 3' 1X 94 C 30" C 30" 30-35X 70 C 2' 70 C 10' 1X 25 C Soak (4) PCR Product Detection Mix 10 µl PCR product with 2 µl 6X loading buffer. Apply each to an agarose gel. Electrophoresis and photograph. Analyze the results and determine the optimal MPCR condition for a specific MPCR application. Each individual MPCR Buffer can also be purchased from MBI. 5

6 VI. Product: EZ-GENXpress TM Normalized Gene Expression Detection Kit (Rat) Description The Normalized Gene Expression Detection Kit is designed for optimization of co-amplification of any specific Rat genes with a specially-designed GAPDH primers. The GAPDH primers have a Tm of 55 o C and can generate a 532 bp PCR products using Rat cdna. The 6 MPCR Buffers provided in the Kit ensure that the MPCR be optimized in hours. The GC-Normalizer TM is used to facilitate DNA strand separation of high GC template. Virtually any human genes can be co-amplified with GAPDH gene under this system as long as they have proper Tm, no serious 3'-end overlap, and don't generate same size of products as GAPDH primers do. Cat. No. EP (GEN-3001) Rxns. 20 Storage Conditions: -20 o C Cat. No. Component Quantity MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl MOB X MPCR Buffer µl GCG-0001 GC-Normalizer TM 1.2 ml GCG-0002 DMSO 0.4 ml GAP-1201/2 GAPDH Primers (10X) 100 µl NTP-0001 dntps (2.5 mm each) 80 µl Sterile ddh 2 O 2.0 ml Instruction Manual 1 copy MPCR Optimization (1) Taq DNA polymerase and its derivative from Perkin Elmer is highly recommended for MPCR. NOTE: SPIN ALL TUBES BEFORE USE!! (2) Reaction Mixture Preparation I: Set up six PCR reactions with the test sample and MPCR Buffer 1 to Buffer 6 (Pink-Capped). If one or more the six buffers provide satisfactory results, there is no need to proceed further. II: If amplify high GC sequences, add proper amounts of DMSO and GC-Normalizer TM into PCR mixture. III: EDTA concentration in test sample must not exceed 0.5 mm because Mg ++ concentration in MPCR Buffers is limited to certain ranges, or add more Mg ++ into PCR mixture to compensate for EDTA. We strongly recommend to run a MPCR reaction with the positive control provided in the Kit. Since the MPCR DNA polymerase needed in each reaction is in a very small volume, it is recommended that all of the PCR components be premixed in a sufficient quantity for daily needs and then dispensed into individual reaction vials to achieve more accurate measurement. Hot start PCR is highly recommended. Per assay Add in order Distilled water 25.5 µl 10X MPCR Buffer 5.0 µl GAPDH Primers (10X) 5.0 µl Specifc Gene Primers (10X) 5.0 µl Taq DNA Polymerase (5 U/ µl) 0.5 µl cdna ( µg/µl) 5.0 µl dntp mixture (2.5 mm each) 4.0 µl DMSO GC-Normalizer TM Mineral oil 50.0 µl (Optional) (3) PCR thermocycle profile: Reaction profiles will need to be optimized according to the machine type and needs of user. A time-temperature profile for the positive control PCR optimized for Perkin Elmer machine type 480 or 9600 is provided as follows: Temperature Time Cycles 94 C 3' 1X 94 C 30" C 30" 30-35X 70 C 2' 70 C 10' 1X 20 C Soak (4) PCR Product Detection Mix 10 µl PCR product with 2 µl 6X loading buffer. Apply each to an agarose gel. Electrophoresis and photograph. Analyze the results and determine the optimal MPCR condition for a specific MPCR application. Each individual MPCR Buffer can also be purchased from MBI. 6

7 VII: Additional Protocol A. RT Protocol The isolation of undegraded, intact RNA is an essential prerequisite for successful first strand synthesis and PCR* amplification. Care should be taken to avoid RNase contamination of buffers and containers used for RNA work by pretreating with DEPC, autoclaving, and baking. Always wear sterile gloves when handling all reagents. 1. Equilibrate a water bath to 42 C, one to 70 C and another to 95 C. 2. On ice, pipet 1-2 ug mrna or 10 ug total RNA dissolved in pure water or 2 ul control GAPDH RNA from kit into a RNase free reaction vial. We strongly recommend to include a positive control reaction at the same time when you set up a RT-PCR at least for first time. 3. Add sterile water to a final volume of 14.5 ul. 4. Add 4 ul random hexamer (50 um) or Oligo(dT) (50 um), incubate at 70 C for 5 minutes and quickly chilled on ice. You may run both hexamer and Oligo(dT) RT reaction at same time. 5. Set up RT by adding following reagents in order: RNAsin 5x RT buffer dntps MMLV RT Per reaction 0.5 ul 10 ul 20 ul 1 ul 6. Incubate at 42 C or 37 C for 60 minutes, heat RT mixture at 95 C for 10 minutes and quickly chill on ice. (This RT mixture can be directly used in PCR reaction, however RT itself may interfere with PCR 5.) 7. Do phenol (ph 8.0) extraction once and phenol/chloroform extraction once of above RT reaction mixture. Take aqueous phase, add 10% volume of 4 M potassium acetate and equal volume ethanol, mix well and put at -70 C for 1 hour or at -20 C overnight. Pellet DNA by spin at maximum speed for 10 minutes. Resuspend the pellet into ul water or TE buffer. 8. Use 5 ul cdna in standard PCR reaction for most of genes. More or less cdna may be used in PCR depending on copy number of the specifc gene. B. Normalizing gene expression using house-keeping gene controls The RT-MPCR provides an accurate way to detect relative genes expression as it amplifies all genes in same condition. The variations in RNA isolation, initial quantitation errors, and tube-to-tube variation in RT and PCR can be compensated by comparing amplification efficiency of samples' cdna with one of house-keeping genes such as GAPDH (multiplex PCR, MPCR) and subsequently using proper amount of cdna in each MPCR. VIII: REFERENCES 1. Chamberlain, J.S. et al., In: The polymerase chain reaction. Mullis K, Ferre F and Gibbs R, eds. Birkhauser Boston Press, 38-46, Baskaran, N et al., 1996 Genome Research 6: Chou, Q et al., 1992 Nucl. Acids Res. 20, Chou, Q et al., 1992 Nucl. Acids Res. 20, Chumakov, K.M. 1994, RT can inhibit PCR and stimulate primer-dimer formation. PCR Methods and Applications. 4: