RealHelix TM qrt-pcr Kit [Intercalator type]

Transcription

1 RealHelix TM qrt-pcr Kit [Intercalator type] CERTIFICATE OF ANALYSIS (1603-V01R03) Kit contents RealHelix TM qrt-pcr Kit [Intercalator type] Cat. No. QRT-S100 (100 rxns) QRT-S500 (500 rxns) qrt-pcr Enzyme Mix [S] 0.2 ml 0.2 ml x 5ea qrt-pcr 2x Buffer Mix [S] 1.25 ml x 2ea 1.25 ml x 10ea ROX Passive Reference Dye 0.1 ml 0.1 ml x 5ea Certificate Analysis 1 ea 1 ea Description The RealHelix TM qrt-pcr kit is a convenient and reliable one-step quantitative RT-PCR kit. Both cdna synthesis and PCR reactions are performed in a single tube using sequence-specific primers corresponding to the target RNAs from total RNA or mrna. The Enzyme Mix in this kit is an optimized blend of HelixCript TM Thermo Reverse Transcriptase and a specially designed automatic hot-start version of PCR enzyme, and ensures reliable results in terms of sensitivity, accuracy and specificity. The 2x Buffer Mix contains all of the required components including optimized buffer components, Mg 2+, dntps, and RNase inhibitor. Two types of qrt-pcr kits are available. qrt-pcr [Intercalator type] is dedicated for SYBR Green detection, and qrt-pcr [Probe type] is for the detection using sequence-specific probes. ROX passive reference dye is also provided with separated tube for normalization of the fluorescence signal. Application Detection of a target gene transcript from RNA in one-tube format Absolute and relative quantification analysis of RNA transcription level Molecular diagnostics for RNA virus Store : -20 qrt-pcr 2x Buffer Mix [S] and ROX Passive Reference Dye should be stored in a dark Quality control assay data Functional analysis The activity for cdna synthesis and quantitative PCR of target transcript using RealHelix qrt-pcr Kit was evaluated by real-time PCR using the 10-fold serial-diluted total RNA isolated from human blood and betaglobin gene-specific primer set with ABI 7900HT fast-real-time PCR system(applied Biosystems, USA) and Rotor- Gene Q instrument (QIAGEN, Germany). Quality authorized by Youn Taek Go

2 qrt-pcr Kit [Intercalator type] Protocol 1. Program a real-time PCR instrument as follows in order to synthesize cdna and PCR amplification. The excitation and emission maxima of the included SYBR Green I are at 494 nm and 521 nm, respectively. <2-step cycling protocol> If annealing temperature(at) of primers used in real-time RT-PCR is between 58 and 62, thermo cycling can be performed using 2-step cycling protocol for PCR Amplification Step as follow. Step Condition Cycle(s) cdna Synthesis 50 for 10 ~ 40 min 1 PCR Enzyme activation 95 for 12 ~ 15 min 1 PCR Amplification Denaturation Annealing & extension 95 for 20 sec 60 for 60 sec Collect the fluorescence data For melting curve analysis of products, refer to the instrument documentation. 40 <3-step cycling protocol> If annealing temperature(at) of primers used in real-time RT-PCR is under than 58 or above 62, thermo cycling can be performed using 3-step cycling protocol for PCR Amplification Step as follow. Step Condition Cycle(s) cdna Synthesis 50 for 10 ~ 40 min 1 PCR Enzyme activation 95 for 12 ~ 15 min 1 Denaturation 95 for 20 sec PCR Amplification Annealing Eextension AT for 30 sec 72 for 30 sec Collect the fluorescence data 40 For melting curve analysis of products, refer to the instrument documentation.

3 qrt-pcr Kit [Intercalator type] 2. Add following components for a single 50 μl reaction volume.* Components RNA Template (1 ng ~ 1 μg)** 2x qrt-pcr Buffer Mix [S] qrt-pcr Enzyme Mix [S] Forward primer (10 pmoles/μl) Reverse primer (10 pmoles/μl) ROX Passive Reference Dye (Optional) DEPC-treated water Volumes X μl 25 μl 1 or 0.1 μl*** Adjust to final 50 μl * For multiple reactions, prepare a master mix by adding the required volumes of each above components (except the RNA template) and dispense appropriate volumes into PCR tubes or plates. * The reaction volume for a reaction could be adjusted according to the recommendations for the instruments. ** Recommended amount of RNA : Total RNA, 10 ng ~ 1 μg, Enriched mrna, 1 ng ~ 500 ng *** See the following table for amount or concentration of ROX Passive Reference Dye for your specific instrument. Instruments Amount of ROX per 50 μl rxn Final ROX Conc. ABI 7000, 7300, 7700, 7900HT, 7900HT Fast, StepOne, StepOnePlus ABI 7500, 7500 Fast. Stratagene Mx3000, Mx3005P, Mx4000 Bio-Rad icycler iq, iq5 and MyiQ, Opticon, CFX 96, CFX 384 Roche LightCycler 480, LightCycler 2.0 Qiagen Rotor-Gene Q Eppendorf MasterCycler ep realplex Cepheid Smart Cycler 1 μl 500 nm 0.1 μl 50 nm Not required Not required To accurately pipette 0.1 μl per reaction, use 1 μl of 10 fold diluted ROX dye. The diluted ROX dye should be prepared immediately before use. 3. Gently mix and immediately centrifuge the reaction mix. 4. Perform the Real-time PCR.

4 qrt-pcr Kit [Intercalator type] Products Cat. No. Products Size QRT-S100 RealHelix qrt-pcr Kit [Intercalator type] 100 rxns QRT-S500 RealHelix qrt-pcr Kit [Intercalator type] 500 rxns

5 RealHelix TM qrt-pcr Kit [Probe type] CERTIFICATE OF ANALYSIS (1603-V01R03) Kit contents RealHelix TM qrt-pcr Kit [Probe type] Cat. No. QRT-P100 (100 rxns) QRT-P500 (500 rxns) qrt-pcr Enzyme Mix [P] 0.2 ml 0.2 ml x 5ea qrt-pcr 2x Buffer Mix [P] 1.25 ml x 2ea 1.25 ml x 10ea ROX Passive Reference Dye 0.1 ml 0.1 ml x 5ea Certificate Analysis 1 ea 1 ea Description The RealHelix TM qrt-pcr kit is a convenient and reliable one-step quantitative RT-PCR kit. Both cdna synthesis and PCR reactions are performed in a single tube using sequence-specific primers corresponding to the target RNAs from total RNA or mrna. The Enzyme Mix in this kit is an optimized blend of HelixCript TM Thermo Reverse Transcriptase and a specially designed automatic hot-start version of PCR enzyme, and ensures reliable results in terms of sensitivity, accuracy and specificity. The 2x Buffer Mix contains all of the required components including optimized buffer components, Mg 2+, dntps, and RNase inhibitor. Two types of qrt-pcr kits are available. qrt-pcr [Intercalator type] is dedicated for SYBR Green detection, and qrt-pcr [Probe type] is for the detection using sequence-specific probes. ROX passive reference dye is also provided with separated tube for normalization of the fluorescence signal. Application Detection of a target gene transcript from RNA in one-tube format Absolute and relative quantification analysis of RNA transcription level Molecular diagnostics for RNA virus Store : -20 ROX Passive Reference Dye should be stored in a dark. Quality control assay data Functional analysis The activity for cdna synthesis and quantitative PCR of target transcript using RealHelix qrt-pcr Kit was evaluated by real-time PCR using the 10-fold serial-diluted total RNA isolated from human blood and betaglobin gene-specific primer set with ABI 7900HT fast-real-time PCR system(applied Biosystems, USA) and Rotor- Gene Q instrument (QIAGEN, Germany). Quality authorized by Youn Taek Go

6 qrt-pcr Kit [Probe type] Protocol 1. Program a real-time PCR instrument as follows in order to synthesize cdna and PCR amplification. Set up the excitation and emission maxima suitable to your fluorescent probe chemistry. <2-step cycling protocol> If annealing temperature(at) of primers used in real-time RT-PCR is between 58 and 62, thermo cycling can be performed using 2-step cycling protocol for PCR Amplification Step as follow. Step Condition Cycle(s) cdna Synthesis 50 for 10 ~ 40 min 1 PCR Enzyme activation 95 for 12 ~ 15 min 1 PCR Amplification Denaturation Annealing & extension 95 for 20 sec 60 for 60 sec Collect the fluorescence data For melting curve analysis of products, refer to the instrument documentation. 40 <3-step cycling protocol> If annealing temperature(at) of primers used in real-time RT-PCR is under than 58 or above 62, thermo cycling can be performed using 3-step cycling protocol for PCR Amplification Step as follow. Step Condition Cycle(s) cdna Synthesis 50 for 10 ~ 40 min 1 PCR Enzyme activation 95 for 12 ~ 15 min 1 Denaturation 95 for 20 sec PCR Annealing AT for 30 sec Amplification 72 for 30 sec Eextension Collect the fluorescence data For melting curve analysis of products, refer to the instrument documentation. 40 Guideline for designing a dual labeled probe - Check that the probe sequence contains more C residues than G residues - Do not put a G on 5 -end. - The optimal probe T m should be ~ 10 higher than primers

7 qrt-pcr Kit [Probe type] 2. Add following components for a single 50 μl reaction volume.* Components RNA Template (1 ng ~ 1 μg)** 2x qrt-pcr Buffer Mix [P] qrt-pcr Enzyme Mix [P] Forward primer (10 pmoles/μl) Reverse primer (10 pmoles/μl) Fluorescent Probe*** ROX Passive Reference Dye (Optional) DEPC-treated water Volumes X μl 25 μl Variable 1 or 0.1 μl**** Adjust to final 50 μl * For multiple reactions, prepare a master mix by adding the required volumes of each above components (except the RNA template) and dispense appropriate volumes into PCR tubes or plates. * The reaction volume for a reaction could be adjusted according to the recommendations for the instruments. ** Recommended amount of RNA : Total RNA, 10 ng ~ 1 μg, Enriched mrna, 1 ng ~ 500 ng *** qrt-pcr Kit [Probe type] can be used with various types of fluorescent probes including TaqMan probe and Molecular Beacons probe, etc. *** See the following table for amount or concentration of ROX Passive Reference Dye for your specific instrument. Instruments Amount of ROX per 50 μl rxn Final ROX Conc. ABI 7000, 7300, 7700, 7900HT, 7900HT Fast, StepOne, StepOnePlus ABI 7500, 7500 Fast. Stratagene Mx3000, Mx3005P, Mx4000 Bio-Rad icycler iq, iq5 and MyiQ, Opticon, CFX 96, CFX 384 Roche LightCycler 480, LightCycler 2.0 Qiagen Rotor-Gene Q Eppendorf MasterCycler ep realplex Cepheid Smart Cycler 1 μl 500 nm 0.1 μl 50 nm Not required Not required To accurately pipette 0.1 μl per reaction, use 1 μl of 10 fold diluted ROX dye. The diluted ROX dye should be prepared immediately before use. 3. Gently mix and immediately centrifuge the reaction mix. 4. Perform the Real-time PCR.

8 qrt-pcr Kit [Probe type] Products Cat. No. Products Size QRT-P100 RealHelix qrt-pcr Kit [Probe type] 100 rxns QRT-P500 RealHelix qrt-pcr Kit [Probe type] 500 rxns