new HCK-M150G - Maxim Biotech, Inc.
Contents
1. 2 IL 1 and TNF are rapidly produced by monocytes and macrophages in response to a number of stimuli such as endotoxins muramyl dipeptides lectins immune complexes and other noxious agents Of these bac teria endotoxin has frequently been used both in vivo and in vitro to stimulate the production of IL 1 and TNF and to study their activity IL 6 is produced by a wide variety of lymphoid and non lymphoid cells both constitutively and in response to many stimuli including other cytokines For ex ample both IL 1 and TNF are potent inducers of IL 6 and once IL 6 is induced cytokines may be distributed to a large number of activity sites via the circulation system Thus their activity may be represented in both local and systemic inflammatory events Inflammatory cytokines can be divided into two groups those involved in acute inflammation and those re sponsible for chronic inflammation Cytokines in acute in flammation include IL 1 TNF alpha IL 6 IL 11 IL 8 and other chemokines G CSF and GM CSF The cytokines in chronic inflammation can be subdivided into cytokines me diating humoral responses such as IL 4 IL 5 IL 6 IL 7 and IL 13 and those mediating cellular responses such as IL 1 IL 2 IL 3 IL 4 IL 7 IL 9 IL 10 IL 12 interferons transforming growth factor s and tumor necrosis factor al pha Some cytokines such as IL 1 significantly contribute to both acute and chronic inflammation 3 4 While the acute production2. of these cytokines is beneficial excessive or sustained production can be deleterious and result in immunopathology The most notable of these recent discoveries is that certain chemokine receptors function as co receptors for HIV 1 Moreover mutations in these receptors can result in host resistance to infection and also affect the progression of disease 9 10 11 For example monocyte chemotatic protein MCP 1 RANTES macrophage inflammatory pro tein MIP 1 alpha MIP 1 beta and Interleukin 8 IL 8 have been found to be more highly expressed in HIV associated dementia 5 In addition chemokines are involved in the migration of leukocytes and have been implicated in sev eral inflammatory diseases of the central nervous system Expression of a variety of checmokines including MCP 1 beta RANTES MIP 1 alpha and IL 8 and receptors in cluding CXCR 4 CCR 1 CCR 3 and CCR 5 have been shown to be increased in HIV encephalitis brain tissue particularly in areas of neuroglial reaction Their expression pattern supported their involvement in the recruitment of inflam matory infiltrates and formation of microglial nodules Ad ditionally presence of chemokine receptors on neurons may be involved in the pathogenesis of neurologic damage in AIDS patients 6 Analysis of the temporal and spatial distribution of RNA expression provides researchers with important clues about the function of apoptosis regulating genes in their own sy
3. 780 Dubuque Avenue So San Francisco CA 94080 U S A Tel 800 989 6296 Fax 650 871 2857 http www maximbio com E mail mbi maximbio com MPCR Kit for Mouse Chemokine Genes Set 1 Cat No MP 70068 50 reactions Cat No MP 70070 100 reactions INSTRUCTION MANUAL ID M10087 Revised August 8 2002 These products are designed and sold for use in the Multiplex PCR MPCR covered by patent 5 582 989 Use of the MPCR process requires a license A limited non automated research field license under the patent to use only this amount of the product to practice the MPCR process is conveyed to the purchaser by the purchase of this product The Polymerase Chain Reaction PCR process is 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 negotiating a license with Perkin Elmer This product is intended for research use only and not for diagnostic purposes INTRODUCTION Cytokines play important roles in orchestrating and controlling inflammation and sepsis process The cytokines IL 1 IL 6 and TNF are sometimes referred to as the in flammatory triad since they mediate both local and sys temic inflammatory responses which are believed to have survival value 1
4. are obtained when the gels are run slowly at less than 100 volts DAN Marker contains linear double stranded DNA bands of 1 000 900 800 700 600 500 400 300 200 and 100 base pairs bp TROUBLESHOOTING 1 MPCR AMPLIFICATION Observation Possible Cause Recommended Action 1 1 No signal or missing some bands during amplification even using positive control provided in kit 1 1a The annealing temperature in thermocycler is too high 1 1b Dominant primer dimers 1 1a Decrease PCR annealing temperature 3 5 C gradually 1 1b Use any one of Hot Start PCR procedures 1 2 Too many nonspecific bands 1 2a The annealing temperature in the thermocycler is too low 1 2b Pre PCR mispriming 1 2c cDNA is interfering with MPCR 1 2a Increase PCR annealing temperature 3 5 C gradually 1 2b Use any one of Hot Start PCR procedures 1 2c Clean cDNA with Phenol Chloroform 1 2d Use Maxim s 3M MPCR Kit 1 3 No difference in gene expression among treatments 1 3a PCR amplification of this specific gene has passed the exponential phase 1 3b Variation in sample preparation RT reaction and amounts of input cDNA 1 3a Decrease PCR cycle number or decrease the input cDNA 1 3b Run a parallel PCR with a house keeping gene to eliminate variables PRECAUTIONS AND STORAGE Storage 1 Store all MPCR Kit components at 20 C Under thes
5. e conditions components of the kit are stable for 1 year 2 Isolate the kits from any sources of contaminating DNA especially amplified PCR product 3 Do not mix MPCR kit components that are from different lots Each lot is optimized individually Beutler B et al 1989 Annu Rev Immunol 7 625 Van Snick J 1990 Annu Rev Immunol 8 253 Schrader J W 1986 Annu Rev Immunol 4 205 Oppenheim J J et al 1991 Annu Rev Immunol 9 617 Kelder W et al 1998 Ann Neurol 44 831 835 Sanders V J et al 1998 AIDS 18 1021 1026 Hayashi K Orita M Suzuki Y amp Sekiya T 1989 Nucleic Acids Res 17 3605 Landgraf A Reckmann B amp Pingoud A 1991 Analytical Biochemistry 193 231 Bloch W 1991 Biochemistry 30 2735 OANDUARWNEFE
6. ld If greater discrimination is necessary several methods are available The simplest procedure is to add a radioactively labeled dNTP to the PCR reaction After gel analysis the band may be excised and counted in a scintillation counter Alternatively the gel may be dried and an autoradio gram may be generated which can be scanned in a densitometer Another method is to label the 5 end of one or both of the primers with 2P which is incorporated into the PCR products and then assayed for radioactivity 7 Southern blot hybridization with synthetic DNA probes may also be performed to verify and quantitate PCR gener ated products either by densitometry of an autoradiogram or by excising and counting the signal from a hybridization membrane This method also quantitates only the target product without interference from nontarget products or primer generated artifacts II Non Radioactive Quantitation Nonradioactive quantitation methods include the use of biotinylated or digoxygenin labeled primers in conjunc tion with the appropriate detection methods 8 use of a bioanalyzer or WAVE For an in depth discussion of the vari ous methods of PCR product quantitation refer to the re view article by Bloch 9 In addition to the above methods several companies now offer gel video systems which can scan and quantitate EtBr stained gel bands in much the same way a densitom eter does Lab on a chip BioAnalyzer CE HPLC and WAVE
7. may also be used to analyze MPCR products and quantitate simultaneously COMPARISON OF MPCR WITH RPA MPCR Multiplex Polymerase Chain Reaction Non isotope method with high sensitivity 0 1 1yg total RNA per MPCR Whole process takes only a few hours Detect Multiple Genes Simultaneously amp Quantitatively Signal can be quantified directly from gel if isotope is included in MPCR Additional techniques can be used to quantify MPCR product using Bioanalyzer HPLC and WAVE y Non specific products can be eliminated by using probes and southern hybridization y Ready to use RPA RNase Protection Assay Isotope or Non Isotope methods 1 20 ug total RNA per RPA assay Whole process takes two days Detect Multiple Genes Simultaneously amp Quantitatively al Ala TA Signal can be quantified directly from gel y Non specific signal can be generated by either low stringent conditions or high secondary structure template y Make own hot RNA probes MPCR KIT DESCRIPTION MPCR Amplification Kits include all necessary MPCR amplification reagents with the exception of Taq Polymerase These kits have been designed to direct the simultaneous amplification of specific regions of human DNA MPCR Kits come in two quantities e 50X 50uL reaction kits e 100X 50uL reaction kits Each kit offers Maxim s optimal primer buffer system which will enhance amplification specificity Figure 1 shows quality co
8. mer 50 mM or Oligo dT 50 mM NOTE The hexamer and Oligo dT RT reactions may be run simultaneously 6 Incubate tube s at 70 C for 5 minutes and quickly chill on ice 7 Begin your RT reaction by adding the following reagents to your hexamer or Oligo mixture 8 Incubate the RT mixture at 37 C for 60 minutes 9 Then heat RT mixture at 95 C for 10 minutes and quickly chill on ice 10 Add another 50 pl water or 0 1X TE buffer 11 2 5 ul of above cDNA is sufficient for most genes in a standard MPCR reaction However more or less DNA may be needed in PCR depending on the copy number of the specific gene PCR Protocol 1 Taq DNA polymerase from Perkin Elmer or its derivatives are highly recommended for MPCR Ampli Taq Gold however is not recommended because its own optimal buffer system is required 2 Reaction Mixture Preparation A Set up MPCR reactions with the test samples and MPCR buffers provided in the MPCR kit according to the table on the next page PROCEDURE Fi 32P dNTPs may be used here to achieve higher sensitivity and better quantitation 5 10 uCi a P dCTP 3000 Ci mmole should be used here per MPCR Keep final dNTPs concentration same as without 32P dNTPs B EDTA concentration in test sample must not exceed 0 5 mM because Mg concentration in MPCR Buffers is limited to certain ranges Additional Mg may be added to the PCR mixture to compensate for EDTA We strongly recommend running a
9. n 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 This will help you to achieve more accurate measure ments 3 PCR thermocycle profile Reaction profiles will need to be optimized according to the machine type and needs of user Please take note that temperature variations occur between different thermocyclers therefore the annealing temperature in the sample profile below is given as a range It will be necessary to determine the optimal temperature for your individual thermocycler An example of a time temperature profile for the positive control PCR reaction optimized for Perkin Elmer machine types 480 2400 and 9600 is provided below Note A 2 step PCR thermocycle profile was found to be more effective than a 3 step PCR thermocycle profile for MPCR amplification For 2 step PCR use 94 95 C for denaturation and 58 60 C for annealing and extension The 72 C step is omitted 4 Agarose Gel Electrophoresis To fractionate the MPCR DNA product electrophoretically mix 10ul of the MPCR product with 2ul 6X loading buffer Run the total 12ul alongside 10 ul of DNA marker from the MPCR kit on a 2 agarose gel containing 0 5 mg ml ethidium bromide Electrophorese and photograph Hint Best results
10. nt Amount mCK1G B001 2X mCK1iG MPCR Buffer 1250 ul X2 containing chemicals enhancer stabilizer and dNTPs mCK1G CO001 10X mCK1G MPCR Pos Control 50ul X2 mCK1G P001 10X mCK1G MPCR Primers 250ul X2 MRB 0014 DNA M W Marker 100bp Ladder 100 ul X2 MRB 0011P ddH 0 DNase free 2 0 ml X2 Instruction Manual REAGENTS FOR AT LEAST 15 SECONDS BEFORE USING NOTE SPIN ALL TUBES BEFORE USING AND VORTEX ALL PROCEDURE 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 reagents Use cDNA derived from 10 cells 1ug cDNA and apply them to each MPCR reaction 1 Prepare total RNA mRNA or use the control GAPDH RNA which is provided in Maxim s MPCR kit NOTE It is best to use cDNA derived from 0 5 1 x 10 cells 0 5 1ug cDNA derived from RNA for each MPCR reaction 2 Equilibrate 3 water baths 37 C 70 C and 95 C 3 On ice pipet 1 2 ug MRNA or 10 ug total RNA from 105 cells dissolved in pure water or 2 ul control GAPDH RNA into a RNAase free reaction vial We strongly recommend including a positive control reaction when setting up an RT PCR reaction for the first time 4 Add sterile water to a final volume of 14 5 ul 5 Add 4 ul random hexa
11. ntrol MPCR results obtained by following MPCR kit manual using different concentrations of positive control For optimal results please read and follow the in structions in this manual carefully If you have any ques tions please contact Maxim Biotech Customer Service at 650 871 1919 Figure 1 M 1 2 3 4 5 6 Lane N PCR using mCK1G Primers without positive Negative Lane 1 PCR using mCK1G Primers with 1X positive Lane 2 PCR using Mouse GAPDH Primers Lane 3 PCR using Mouse MCP 1 Primers Lane 4 PCR using Mouse MCP 2 Primers Lane 5 PCR using Mouse IP 10 Primers Lane 6 PCR using Mouse Rantes Primers Lane M DNA M W Marker MPCR PRIMER INFORMATION Product Gene 5 3 Tm Amplicon Accession Intron Genomic Code Size No Span Size mCK1G IP10 Mouse IP 10 68 C 68 C 198bp NM_0212714 yes 391bp mCK1G RAN Mouse Rantes 68 C 69 C 176bp NM_013653 no 176bp mCK1G MCP2 Mouse MCP2 67 C 67 C 233bp NM_021443 yes 1336bp mCK1G MCP1 Mouse MCP1 67 C 67 C 275bp NM_011333 yes 1344bp mCK1G GAP Mouse GAPDH 70 C 67 C 532bp M32599 no 532bp KIT COMPONENTS Product Code Kit Component Amount mCK1G B001 2X mCKiG MPCR Buffer 1250 ul containing chemicals enhancer stabilizer and dNTPs mCK1G C001 10X mCK1G MPCR Pos Control 50ul mCK1G P001 10X mCK1iG MPCR Primers 250ul MRB 0014 DNA M W Marker 100bp Ladder 100 ul MRB 0011P ddH 0 DNase free 2 0 ml Instruction Manual Product Code Kit Compone
12. stems Northern Blot and RNase Protection Assay are the most widely used procedures for determining the abundance of a specific mRNA in a total or poly A RNA sample RT MPCR provides an alternate and accurate method to detect multiple gene expression by amplifying all the genes under the same conditions 8 9 10 Variations in RNA isolation initial quantitation errors or tube to tube varia tions in RT and PCR can be compensated by including a house keeping gene such as GAPDH in MPCR Alterna tively a parallel RT PCR using the same cDNA PCR condi tions and primers for one of house keeping genes may be run to offset any variations Differences in gene expression can be determined by normalizing its expression against GAPDH expression Maxim s Mouse Chemokine MPCR kits have been de signed to detect the expression of Mouse IP 10 Rantes MCP 1 MCP 2 and GAPDH genes The PCR primers have similar Tm and no obvious 3 end overlap to enhance mul tiple amplification in a single tube The kit will yield 532 bp GAPDH 198 bp IP 10 176 bp Rantes 233 bp MCP 1 and 275 bp MCP 2 PCR products with RNA from human cells or positive controls from kit The gene expression of these genes can be analyzed and compared with GAPDH gene expression PCR PRODUCT QUANTITATION I Radioactive Quantitation In our experience visual inspection of an EtBr stained agarose gel is sensitive and precise enough to detect changes as low as two fo
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