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RT-PCR kits

Product Description

Real-time quantitative PCR (RT-qPCR) is an innovative and reliable technique for quantitative analysis of gene expression, mutation detection, allele discrimination and single nucleotide polymorphisms (SNP) genotyping.

  • Description
  • Experimental
  • Usage
  • Application
  • Ordering

Fluorescence Quantitative PCR related Product
Real Time Quantitative PCR
Real-time quantitative PCR (RT-qPCR) is an innovative and reliable technique for quantitative analysis of gene expression, mutation detection, allele discrimination and single nucleotide polymorphisms (SNP) genotyping. Real-time quantitative PCR was developed to overcome the basic weakness of the classical PCR technology: whereas PCR can indicate the presence of a particular nucleic acid in a sample after a completed PCR amplification, it cannot directly quantify the amount of amplification.
Real-time quantitative PCR systems determine the amount of amplified product by measuring the quantity of a fluorescent dye. This dye, upon excitation, emits a light or signal that increases in intensity in direct proportion to the amount of amplified PCR product and, therefore, quantifies the amount of target DNA. The assays can be directly performed in a sealed tube without any purification or separation step, avoiding all risks of contamination and post-PCR handling.
Real-time quantitative PCR systems also enable mutation detection via melting-curve analysis, as each double-stranded DNA product has its own specific melting temperature (Tm). Melting-curve data enables the researcher to differentiate between specific PCR products and non-specific PCR products, such as primer-dimers.

 Sequence-specific Probes and Non-Sequence-Specific Probes
Detection by real-time quantitative PCR can be sequence-specific or non-sequence specific, and the method chosen will affect the performance of your real-time quantitative PCR results. For instance, SYBR® Green 1, a non-sequence-specific reagent, can bind to any double-stranded DNA including primer-dimers and other non-specific reaction products, and so may lead to an over-estimation of the amount of target DNA. However, sequence-specific probes, such as Taqman probe, dual-labeled fluorescence probe, or Molecular Beacons, bind only to the target template and therefore achieve accurate and reliable quantification of the amount of target DNA.

Design and Synthesis Service of RT-PCR Primers
 GenePharma provides design of primer or probe for free.
We design primers by using of optimized primer design techniques and latest database resources, simultaneously utilizing all kinds of bioinformatics knowledge and skills to select the best primers. You may get the best experimental results by using of primers and RT-PCR reagents provided by our company in our recommended standard reaction conditions.

 Service for primer design has following characteristics:

  1. Not easy to form primer-dimer;
  2. Primers are normally designed crossing exon junction, which makes it hard to amplify genomic DNA;
  3. The sequence of primer does not contain known SNP site (Confirmed by dbSNP);
  4. The specificity is confirmed through homology search;
  5. Choosing the design region under experimental purposes;
  6. Normally, 1 - 3 pairs of corresponding primers are provided for one target gene.
  7. Design according to the full sequence of target gene;
  8. Design among the 1,500 bases at 5’ end;
  9. Design among the 1,500 bases at 3’ end.

Customer must provide the following information: 

  1. Biology species information (such as Human, Mouse, Rat);
  2. Target gene information (GeneBank Acc, GeneID, key word);
  3. Primer design region (full sequence, 5 ' end sequence, 3 ' end sequence);
  4. Primers, including SYBR Green I, Taqman probes and molecular beacons primers.

Cat. No.

Probe Type

Purification

Quantity

Price

Times

5’ reporter

3’ quencher

P-01

FAM

DABCYL

PAGE+HPLC

2 OD

$99

1 week

P-02

FAM

TAMRA

PAGE+HPLC

2 OD

$99

1 week

P-03

FAM

BHQ-1

PAGE+HPLC

2 OD

$108

1 week

P-04

TAMRA

BHQ-2

PAGE+HPLC

2 OD

$108

1 week

P-05

HEX

DABCYL

PAGE+HPLC

2 OD

$133

1 week

P-06

Cy3

DABCYL

PAGE+HPLC

2 OD

$158

1 week

P-07

TET

DABCYL

PAGE+HPLC

2 OD

$116

1 week

P-08

JOE

DABCYL

PAGE+HPLC

2 OD

$133

1 week

P-09

ROX

BHQ-2

PAGE+HPLC

2 OD

$133

1 week

enePharma SYBR Real-Time PCR Kit User Manual

 

 

Overview

 

 

 

 

 

Introduction

The SYBR® Real-Time PCR Kit provides qualified reagents in lyophilized form for the amplification and detection of DNA in quantitative real-time polymerase chain reaction (qPCR). The SuperMix formulation is aliquoted into plate wells or strip wells and then lyophilized for room temperature storage and ease of reaction setup. To perform PCR, simply add water, primers, and template, vortex to dissolve the pellet, and proceed with the reaction.

 

 

 

The system enables highly sensitive detection from as few as 10 copies of a target gene, with a broad dynamic range that supports accurate quantification of high-copy gene from up to 1 μg of DNA:

 

□ 2×SYBR® Green Reaction Mix consists of a proprietary buffer system, SYBR® Green I, MgSO4, dNTPs, and stabilizers. SYBR® Green I is a fluorescent dye that binds directly to double-stranded DNA (dsDNA). In qPCR, as dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and that can be detected using real-time qPCR instruments. SYBR® Green I in this ready to use formulation can quantify as few as 10 copies of a target gene in as little as 1 pg of total DNA or RNA. It has a broad dynamic range of seven orders of magnitude, and is compatible with melting curve analysis.

 

 

 

 

Uses for the
SYBR® Real-Time PCR Kit

Use the One-Step SYBR® Real-Time RT-PCR Kit in your experiments for the following purposes:

  1. To quantify a target gene in DNA, especially the low expression genes.
  2. To analyze the relative expression ratio between a target gene and a housekeeping gene.

 

 

Components of the Kit

 

 

 

 

Introduction

This section provides more information about the reagents supplied in the One-Step SYBR® Real-Time RT-PCR Kit.

 

 

 

 

 

 

 

 

 

SYBR PCR Master Mix
2×Conc.

The 2×SYBR® Green Reaction Mix consists of a proprietary buffer system, SYBR® Green I, MgCl2, dNTPs, and stabilizers. The mix includes 0.2 mM of each dNTP and 3mM MgCl2, and has been confirmed to be work well for most targets in restrict lab experiments. However, the optimal Magnesium concentration may range from 3 to 6 mM, and so if necessary, use the separate tube of 25mM magnesium sulfate to increase the magnesium concentration.

It’s very important for you to store the SYBR PCR Master Mix in the dark.

 

 

Taq DNA polymerase
5U/μl

For most PCR reaction, the final concentration of DNA polymerase was ussually 0.05U/μl per reaction volume.

 

 


Method

 

 

Performing Real-Time PCR

 

 

 

 

Introduction

In SYBR® Green real-time PCR, as dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and that can be detected using real-time qPCR instruments. This section provides guidelines and an example protocol for performing SYBR real-time PCR.

 

 

 

 

Instrument Compatibility

This kit can be used with a variety of real-time instruments, including but not limited to the ABI PRISM 7000, 7700, and 7900HT; the ABI 7300 and 7500 Real-Time PCR Systems; the Bio-Rad iCycler; the Stratagene Mx3000P and Mx4000; the Corbett Research Rotor-Gene 3000; the MJ Research DNA Engine Opticon, Opticon 2, and Chromo 4 Real-Time Detector; and the Cepheid Smart Cycler .Optimal cycling conditions will vary slightly with different instruments.

 

 

 

 

Primer design &
Concentration

Primer selection is one of the most important parameters for qPCR when using a SYBR Green detection system. When designing primers, keep in mind that the amplicon length should be approximately 80–250 bp to optimize the efficiency of qPCR. We strongly recommend using a primer design program such as Oligo 6.0.
Ensure that primers are specific for the target sequence and free of internal secondary structure, and avoid complementation at 3’-ends within each primer and with each other. A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a titration of primer concentrations between 100 and 500 nM.

 

 

 

 

Template
Specifications

The target template for SYBR real-time PCR is plasmid DNA (10 to 107 copies), genomic DNA (100 pg to 1μg), or cDNA (generated from 1 pg to 100 ng of total RNA). For best results, the amplicon size should be limited to 80–250 bp in size.

 

 

 

 

Magnesium Concentration

The 2×SYBR Green Reaction Mix includes magnesium at a final concentration of 5 mM which has been confirmed to be work well for most targets in restrict lab experiments. However, the optimal concentration may range from 3 to 6 mM. If necessary, use the separate tube of 25mM magnesium chloride to increase the magnesium concentration.

 

 

 

 


 

 

 

 

 

2. Three-Step Standard Cycling Program
95℃ for 3 minute hold;
40 cycles of:
95℃, 15 seconds
55℃, 30 seconds
72℃, 30 seconds
Melting Curve Analysis
Program according to instrument instructions.

 

 

 

3. Two-Step Standard Cycling Program

 

95℃ for 3 minute hold;
40 cycles of:
95℃, 15 seconds
60℃, 30 seconds
Melting Curve Analysis
Program according to instrument instructions.

 

 

 

 

Optimal temperatures and incubation times may vary for different target sequences.

 

 

 

 

1. Make sure that all components are at the bottom of the tube/plate; centrifuge briefly if needed.
2. See the table on the previous page for the amount/concentration of ROX to use for your specific instrument.
3. Melting curve analysis can identify the presence of primer dimers by their lower annealing temperature, compared to the amplicon. The presence of primer dimers is not desirable in samples that contain template, as it decreases PCR efficiency and obscures analysis and determination of cycle thresholds.
4. The formation of primer dimers most often occurs in no-template controls, where the polymerase enzyme is essentially idle, and in this case the quantitative analysis of the template samples is not affected.
5. Melting curve analysis of no-template controls can discriminate between primer dimers and spurious amplification due to contaminating nucleic acids in reagent components.

 

 

 

 

 

 

 

World Wide Web

Visit the Invitrogen Web Resource using your World Wide Web browser. At the site, you can:

 

□ Download manuals in Adobe Acrobat (PDF) format

 

□ Explore our catalog with full color graphics

 

□ Get the scoop on our hot new products and special product offers

 

□ Obtain citations for Invitrogen products

 

□ Request catalog and product literature

 

 

 

The Genepharma URL is www.genepharma.com

 

 

 

 

Quality Control

The product is tested functionally by qRT-PCR using total HeLa RNA as template. Kinetic analysis must demonstrate a linear dose response with decreasing target concentration and detection of GAPDH mRNA in 1 pg of total HeLa RNA.

 

 

 

 

Contact Us

For more information or technical assistance, please call, write, fax, or email. Additional international offices are listed on our web page (www.genepharma.com).

 

 

 

Corporate Headquarters:
Genepharma Corporation
1011 Halei Road,
Zhangjiang High-tech District,
ShangHai, CHINA
Tel: 86-21-51320195
Fax: 86-21-51320295
E-mail: service@genepharma.com

 

 

 

 

 

 

 

 


References

Edwards, K., Logan, J., and Saunders, N. (2004) Real-Time PCR: An Essential Guide. Horizon Scientific Press, Wymondham, Norfolk, UK.

 

Wittwer C.T., Herrmann M.G., Moss A.A., and Rasmussen R.P. (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22, 130-138.

 

Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993). Kinetic PCR Analysis: Real-Time Monitoring of DNA Amplification Reactions. Biotechnology 11, 1026-1030.

 

©2005–2006 Genepharma Corporation. All rights reserved. For research use only. Not intended for any animal or human therapeutic or diagnostic use.

 

1. Bastian, I., S. Tam Tam, et al. "Differential expression of microRNA-1 in dorsal root ganglion neurons." Histochemistry and cell biology 135(1): 37-45.

2.Ma, H., Q. Lu, et al. "Functional analysis of the cellulose gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference." Genetics and Molecular Research 10(3): 1931-1941.

3. Zou, Z., L. Wu, et al. "MicroRNA-30a sensitizes tumor cells to cis-platinum via suppressing beclin 1-mediated autophagy." Journal of Biological Chemistry 287(6): 4148-4156.

4. Li, N., J. Cui, et al. "Suppression of type I collagen expression by miR-29b via PI3K, Akt, and Sp1 pathway in Human Tenon’s Fibroblasts." Investigative Ophthalmology & Visual Science 53(3): 1670-1678.

5. Lan, Y., K. Zhao, et al. "Inhibition of porcine hemagglutinating encephalomyelitis virus replication by short hairpin RNAs targeting of the nucleocapsid gene in a porcine kidney cell line." Journal of virological methods.

6.Gao, S., J. Yang, et al. "Pure curcumin decreases the expression of WT1 by upregulation of miR-15a and miR-16-1 in leukemic cells." Journal of Experimental & Clinical Cancer Research 31(1): 27.

7. Zhang, X., Z. Liu, et al. "Neferine, an alkaloid ingredient in lotus seed embryo, inhibits proliferation of human osteosarcoma cells by promoting p38 MAPK-mediated p21 stabilization." European Journal of Pharmacology.

8. Bian, H. B., X. Pan, et al. "Upregulation of microRNA-451 increases cisplatin sensitivity of non-small cell lung cancer cell line (A549)." J Exp Clin Cancer Res 30(1): 20.

9. Sun, K., W. Wang, et al. "MicroRNA-221 inhibits CDKN1C/p57 expression in human colorectal carcinoma." Acta Pharmacologica Sinica 32(3): 375-384.

10. Xu, Z., J. Jiang, et al. "MicroRNA-181 Regulates CARM1 and Histone Aginine Methylation to Promote Differentiation of Human Embryonic Stem Cells." PloS one 8(1): e53146.

Ordering
The first problem we meet for the application of siRNA is to design the sequences. Although there are quite a lot principles for the designing of published sequences and quite a few companies supply on-line design service, up to now, no software can guarantee the efficiency of the oligos designed.
Shanghai GenePharma Co, Ltd. and our collaboration laboratories have been involved in RNAi research for some time. For each specific gene target, if 4 pairs of oligos were designed and synthesized, normally you will have at least one pair showing 70% down regulation.
Our price: 2OD, PAGE purified, $89/pair
*If one single order is less than 5 pairs extra shipping fee of $40 will be charged. Payment terms are 30 days. We accept cheque or wire transfer into our USD account.
Specifications of siRNA Oligos:
Quality Control:
All our siRNA oligos undergo vigorous process monitoring and strict quality control. Length and labeling are systematically controlled by PAGE or mass spectrometry analysis. Quantity is systematically validated by UV absorbance at 260 nm.
Purification: Fully deprotected and desalted.
Purified by PAGE or RP-HPLC upon request
Length: 19 to 23 mers
Bases: RNA (A, C, G or U)
DNA (A, C, G or T)
Backbone: Phosphodiester bond
Labels and modifications: Fluorescein, biotin and phosphate: 3’ or 5’end
Other labels available upon request
Format: Single-strand RNA oligos is delivered in dry form
Storage and stability
Although oligonucleotides are stable in solution at 4°C for up to 2 weeks, we recommend storage should be at -20°C. Repetitive freeze-thaw cycles should be avoided by storing as aliquots. For long-term storage, siRNA oligos should be dried.
Oligonucleotides with fluorescent labels should be protected from light. We guarantee our oligonucleotides for six months, when stored under the above conditions.
Shipment: Shipped by express delivery, dry in individual, transparent tubes.
Oligonucleotide Technical Data Sheet
Oligonucleotides are delivered with an Oligonucleotide Technical Data Sheet, which includes oligonucleotide name, sequence, concentration, size,  purification method.

Services available upon request

  1. Aliquoting
  2. Free design support
  3. Additional services may increase turn-around time

Ordering:Please fill in the following order form and send it to: order@genepharma.com or by fax to:00862151320295 our staff will confirm with you asap.