AmoyDx TM RRM1 Gene Expression Analysis Kit Instructions For Use Instructions Version: B2.1 Date of Revision: April 2012 Store at -20±2 o C -1/5-
Background RRM1 is one of the targets of Gemcitabine, and it has been demonstrated that high RRM1 gene expression is associated with Gemcitibine resistance. In addition, late stage cancer patients with high RRM1 gene expression have a less favorable prognosis than patients with low RRM1 gene expression. The quantitative assessment of RRM1 gene expression has bee used clinically to determine the suitability of Gemcitabine for NSCL cancer patients. Intended Use AmoyDx RRM1 Gene Expression Analysis Kit is intended for research use only. Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 1). Both the target RRM1 gene and the internal reference β-actin gene are indicated by FAM signals. Table 1 Kit Contents Reagents Supplied Size Tube No. RRM1 Taq DNA Ploymerase 45 µl/tube 1 M-MLV Reverse Transcriptase 30 µl/tube 2 Reverse Transcription Reaction Mix 500 µl/tube 3 RRM1 Reaction Mix 1400 µl/tube 4 β-actin Reaction Mix 1400 µl/tube 5 Gene Expression Standard (10 3 Copies/uL) 120 µl/tube 6 Gene Expression Standard (10 4 Copies/uL) 120 µl/tube 7 Gene Expression Standard (10 5 Copies/uL) 120 µl/tube 8 Gene Expression Standard (10 6 Copies/uL) 120 µl/tube 9 Equipment and Reagents Not Supplied With Kit 1. The compatible PCR instruments are: Stratagene Mx3000P, Stratagene Mx3005P, ABI7300, ABI7500, LightCycler480Ⅰ and Ⅱ. 2. Sterile, nuclease-free tubes. 3. Dedicated pipette and filter pipette tips for handling DNA template. 4. Sterile, nuclease-free H2O. Shipping and Storage The kit requires cold-chain-transportation and be stored immediately upon receipt at -20±2 o Cina constant-temperature freezer and protected from light. The shelf-life of the kit is indicated on the package. Specimen Material Human genomic RNA must be extracted from materials prior to use. If it is not used immediately, it should be stored below -70 o C for no more than six months. Good RNA quality is essential and we recommend use of Qiagen RNA extraction kits. The OD value of RNA samples should be measured using the spectrophotometer after extraction. The Thermo Fisher NanoDrop 1000 /2000 spectrophotometer is recommended. The A260/A280 value should be between 1.9 and 2.1. Technological Principles The cdna is obtained from sample RNA through reverse transcription, then the target gene sequence is specifically amplified by novel, proprietary primers. The amount of target cdna is measured after each cycle in the data capture phase using a novel fluorescent probe. Note: Reporter Dye: FAM; Quencher Dye: TAMRA; Passive Reference: NONE. The analysis of the results can be based on either the standard curve method or the Ct comparison method (2 -ΔΔ Ct method). The kit contains standards for the standard curve method. The Ct comparison method requires a user-supplied control or calibrator sample. A word about nomenclature: target gene = RRM1, reference gene = β-actin, test sample = tumor, control or calibrator sample = normal tissue. Protocol -2/5-
1. Reverse Transcription. (1) For each RNA sample, transfer 16 µl Reverse Transcription Reaction Mix and 1 µl M-MLV Reverse Transcriptase to a sterile centrifuge tube; mix well by pipetting gently up and down. (2) Add 3 µl of sample RNA into the appropriate centrifuge tube. The total amount of RNA should be within 10 ng ~ 2 μg. (3) Incubate the tubes at 42 o C for one hour. (4) Heat the tubes at 95 o C for five minutes, then transfer them to ice. The resulting cdna solutions are used for PCR amplification. 2. PCR amplification. 2.1 Standard Curve Method for Analysis of Absolute Gene Expression (1) According to the ratio of 23 µl Reaction Mixture to 0.15 µl RRM1 Taq DNA Polymerase per sample, transfer the appropriate amount of Reaction Mix and Taq DNA Polymerase into a sterile tube. (2) Mix well by pipetting up and down and centrifuge for 15 seconds. (3) Transfer 23 µl of the polymerase solution into PCR reaction wells. (4) Add 2 µl of sample cdna, 2 µl of the various Gene Expression Standards and 2 µl ddh2o (no-template control) solutions to the appropriate PCR reaction wells. (5) An example of a plate layout is shown in Table 2. Table 2. Plate Layout for Standard Curve Method (example for 24 tests/kit) Code 1 2 3 4 5 6 7 8 RRM1 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 RRM1 Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 RRM1 Sample 17 Sample 18 Sample 19 NTC Standard 10 3 Standard 10 4 Standard 10 5 Standard 10 6 β-actin Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 β-actin Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 β-actin Sample 17 Sample 18 Sample 19 NTC Standard 10 3 Standard 10 4 Standard 10 5 Standard 10 6 (6) Seal the PCR tubes. (7) Spin the PCR tubes gently to collect the reagents at the bottom of wells. (8) Place the PCR tubes into the real-time PCR instrument. (9) Carry out real-time PCR using the cycling conditions described in Table 3. Table 3 Cycling Parameters Temperature Time Cycles Stage 1 94 5min 1 Stage 2 60 20s 10 72 20s Stage 3 58 35s Data collection of FAM 35 72 15s 2. 2 Ct Comparison Method. Use this method to compare target gene expression in the test samples to target gene expression in a control, or calibrator, sample. The target gene expression is normalized using the reference gene expression. The control sample might be normal tissue isolated from a region adjacent to test cancer tissue. (1) According to the ratio of 23 µl Reaction Mixture to 0.15 µl RRM1 Taq DNA Polymerase, transfer the appropriate amount of Reaction Mix and Taq DNA Polymerase into a tube. (2) Mix well by pipetting up and down. (3) Centrifuge briefly. (4) Transfer the solution to the PCR reaction wells in the amount of 23 µl per well. -3/5-
(5) Add 2 µl sample cdna, 2 µl control sample and 2 µl ddh2o(no-template control)solutions to the solutions individually. (6) An example of a plate layout is shown in Table 4. Table 4. Plate Layout for Relative Gene Expression (example for 24 tests/kit) Code 1 2 3 4 5 6 7 8 RRM1 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 RRM1 Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 RRM1 Sample 17 Sample 18 Sample 19 Sample 20 Sample 21 Sample 22 Control NTC β-actin Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 β-actin Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 β-actin Sample 17 Sample 18 Sample 19 Sample 20 Sample 21 Sample 22 Reference Sample NTC (7) Seal the PCR tubes. (8) Spin the PCR tubes gently to collect the reagents at the bottom of wells. (9) Place the PCR tubes into the real-time PCR instrument. (10) Carry out real-time PCR using the cycling conditions described in Table 5. Table 5 Cycling Parameters Temperature Time Cycles Stage 1 94 5min 1 Stage 2 60 20s 10 72 20s Stage 3 58 35s Data collection of FAM 35 72 15s Analysis of RT qpcr Data The experiment can be designed to yield the relative amount of target gene expression in tumor tissue compared to control sample. Brief descriptions are provided below. 1. Standard Curve Method The absolute concentration of the target gene can be determined by making a graph with the standards provided in the kit. The expression level of the target gene can then be determined by interpolation (1) Make a standard curve using the RRM1 gene standards and β-actin gene standards. a. X axis: log of the concentration of the standards b. Y axis: Ct values for standards (2) Record C1 value on RRM1 gene standard curve, and the sample C2 value on β-actin standard curve, then obtain the sample s relative ratio = C1/C2. (3) Note: The setting of data analysis: a. User could adjust the Start value, Stop value and Threshold value of Baseline. b. The adjustment range of Start value is 1~10, Stop value is 5~20, Threshold value is 0.01~0.2. c. Optimize the standard curve by adjusting these values so that the correlation coefficient R is in the range of 0.97~ 1.0. d. Choose "Analyze" under the Analysis menu to analyze the data automatically. 2. Ct Comparison Method The formulas used for the ΔΔCt method are given below. (1) Calculate the ΔΔCt value for the gene expression differences between test sample (tumor) and control sample using this formula: -4/5-
Formula 1: ΔΔCt = (Ct RRM1 gene -Ctβ-actin) Test sample -(CtRRM1 gene -Ctβ-actin) Control Sample (2) Calculate the fold difference in gene expression using this formula: Formula 2: 2 ΔΔCt = normalized expression ratio. Warnings and Precautions 1. Please read the instruction carefully and become familiar with all components of the kit prior to use. 2. Do not exchange and mix up the kit contents with different batches. 3. The kit and its contents cannot be resold or modified for resale without the written approval of AmoyDx. 4. Using other sources of reagents is not recommended. Strictly distinguish the reagents from mixed standard to avoid contamination. Otherwise, false positive may be produced. 5. Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended that users have separate, dedicated pipettes and filter pipette tips to add DNA template and during the preparation of reagents. 6. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching. 7. Only trained professionals should use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 8. AmoyDx grants the customer a non-exclusive and non-transferable license to use AmoyDx technologies. 9. AmoyDx assumes no responsibility for any errors that may appear in this document. The information in this document is subject to change. References 1. Rosell R.,et al. 2004. Clin Cancer Ras. 10:1318-25. 2. Bepler G.,et al. 2004. J. Clin. Oncol. 15:1878-85. -5/5-