RT² SYBR^® Green qPCR Mastermixes

For use with RT² Profiler, RT² lncRNA, EpiTect ChIP, and EpiTect Methyl II PCR Arrays, and qPCR Assays

Products

RT² SYBR^® Green qPCR Mastermixes are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

RT² SYBR Green Fluor qPCR Mastermix (24)

Cat. No. / ID: 330513

Contains 24 x 1.35 ml tubes: for 24 x 96-well RT² PCR Arrays, 16 x 384-well RT² PCR Arrays or 2400 x 25 µl reactions or 6000 x10 µl reactions

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RT² SYBR Green qPCR Mastermix (12)

Cat. No. / ID: 330502

Contains 12 x 1.35 ml tubes: for 12 x 96-well RT² PCR Arrays, 8 x 384-well RT² PCR Arrays or 1200 x 25 µl reactions or 3000 x 10 µl reactions

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RT² SYBR Green ROX qPCR Mastermix (24)

Cat. No. / ID: 330523

Contains 24 x 1.35 ml tubes: for 24 x 96-well RT² PCR Arrays, 16 x 384-well RT² PCR Arrays or 2400 x 25 µl reactions or 6000 x 10 µl reactions

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RT² SYBR Green qPCR Mastermix (2)

Cat. No. / ID: 330500

Contains 2 x 1.35 ml tubes: for 2 x 96-well RT² PCR Arrays, 1 x 384-well RT² PCR Arrays or 200 x 25 µl reactions or 400 x 10 µl reactions

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RT² SYBR Green ROX qPCR Mastermix (25 ml)

Cat. No. / ID: 330529

Contains 1 bottle of 25 ml mastermix: for 20 x 96-well RT² PCR Arrays, 12 x 384-well RT² PCR Arrays or 2000 x 25 µl reactions or 5000 x 10 µl reactions

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RT² SYBR Green Fluor qPCR Mastermix (12)

Cat. No. / ID: 330512

Contains 12 x 1.35 ml tubes: for 12 x 96-well RT² PCR Arrays, 8 x 384-well RT² PCR Arrays or 1200 x 25 µl reactions or 3000 x 10 µl reactions

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RT² SYBR Green qPCR Mastermix (24)

Cat. No. / ID: 330503

Contains 24 x 1.35 ml tubes: for 24 x 96-well RT² PCR Arrays, 16 x 384-well RT² PCR Arrays or 2400 x 25 µl reactions or 6000 x 10 µl reactions

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RT² SYBR Green ROX qPCR Mastermix (6)

Cat. No. / ID: 330524

Contains 6 x 1.35 ml tubes: for 6 x 96-well RT² PCR Arrays, 3 x 384-well RT² PCR Array or 600 x 25 µl reactions or 1500 x 10 µl reactions

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RT² SYBR Green Fluor qPCR Mastermix (2)

Cat. No. / ID: 330510

Contains 2 x 1.35 ml tubes: for 2 x 96-well RT² PCR Arrays, 1 x 384-well RT² PCR Array or 200 x 25 µl reactions or 400 x 10 µl reactions

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RT² SYBR Green qPCR Mastermix (25 ml)

Cat. No. / ID: 330509

Contains 1 bottle of 25 ml mastermix: for 20 x 96-well RT² PCR Arrays, 12 x 384-well RT² PCR Arrays or 2000 x 25 µl reactions or 5000 x 10 µl reactions

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RT² SYBR Green ROX qPCR Mastermix (8)

Cat. No. / ID: 330521

Contains 8 x 1.35 ml tubes: for 8 x 96-well RT² PCR Arrays, 4 x 384-well RT² PCR Arrays or 800 x 25 µl reactions or 2000 x 10 µl reactions

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RT² SYBR Green qPCR Mastermix (8)

Cat. No. / ID: 330501

Contains 8 x 1.35 ml tubes: for 8 x 96-well RT² PCR Arrays, 4 x 384-well RT² PCR Arrays or 800 x 25 µl reactions or 2000 x 10 µl reactions

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RT² SYBR Green Fluor qPCR Mastermix (25 ml)

Cat. No. / ID: 330519

Contains 1 bottle of 25 ml mastermix: for 20 x 96-well RT² PCR Arrays, 12 x 384-well RT² PCR Arrays or 2000 x 25 µl reactions or 5000 x 10 µl reactions

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RT² SYBR Green Fluor qPCR Mastermix (6)

Cat. No. / ID: 330514

Contains 6 x 1.35 ml tubes: for 6 x 96-well RT² PCR Arrays, 3 x 384-well RT² PCR Array or 600 x 25 µl reactions or 1500 x 10 µl reactions

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RT² SYBR Green ROX qPCR Mastermix (12)

Cat. No. / ID: 330522

Contains 12 x 1.35 ml tubes: for 12 x 96-well RT² PCR Arrays, 8 x 384-well RT² PCR Arrays or 1200 x 25 µl reactions or 3000 x 10 µl reactions

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RT² SYBR Green Fluor qPCR Mastermix (8)

Cat. No. / ID: 330511

Contains 8 x 1.35 ml tubes: for 8 x 96-well RT² PCR Arrays, 4 x 384-well RT² PCR Arrays or 800 x 25 µl reactions or 2000 x 10 µl reactions

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RT² SYBR Green qPCR Mastermix (6)

Cat. No. / ID: 330504

Contains 6 x 1.35 ml tubes: for 6 x 96-well RT² PCR Arrays, 3 x 384-well RT² PCR Arrays or 600 x 25 µl reactions or 1500 x 10 µl reactions

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RT² SYBR Green ROX qPCR Mastermix (2)

Cat. No. / ID: 330520

Contains 2 x 1.35 ml tubes: for 2 x 96-well RT² PCR Arrays, 1 x 384-well RT² PCR Array or 200 x 25 µl reactions or 400 x 10 µl reactions

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Features

Designed exclusively for use with RT² and EpiTect Methyl II qPCR Assays
Delivers specific amplification with minimal primer-dimers
Available with ROX, fluorescein, or without dye
For use with qPCR instruments under standard cycling conditions

Product Details

RT² SYBR Green qPCR Mastermixes are highly suited for real-time PCR applications using SYBR Green, and are required for guaranteed qPCR performance of the RT² qPCR Primer Assays, RT² Profiler PCR Arrays, RT² lncRNA qPCR Assays, and RT² lncRNA PCR Arrays. This Mastermix should be used in qPCR instruments under standard cycling conditions. RT² SYBR Green qPCR Mastermixes are also suitable for use with EpiTect ChIP and EpiTect Methyl II qPCR Assays and PCR Arrays.

Performance

RT² SYBR Green qPCR Mastermixes contain all of the optimized reagents and buffers needed for SYBR Green based real-time PCR with RT² qPCR Primer Assays, RT² Profiler PCR Arrays, RT² lncRNA qPCR Assays, and RT² lncRNA PCR Arrays on all real-time PCR instruments.

The high-performance real-time PCR enabled by RT² SYBR Green qPCR Mastermixes demonstrates high amplification efficiencies (see figure " High amplification efficiency over a wide dynamic range") and high levels of sensitivity and specificity (see figure " Tighter control of polymerase activity yields greater specificity").

See figures

High amplification efficiency over a wide dynamic range.

Tighter control of polymerase activity yields greater specificity.

Principle

RT² SYBR Green qPCR Mastermixes contain real-time PCR buffer, a high-performance, HotStart DNA Taq polymerase, nucleotides, and SYBR Green dye. Some mastermixes contain either fluorescein or ROX dye for optimization of the instrument optics. The chemically-modified and tightly controlled HotStart enzyme uniquely provides accurate SYBR Green results by preventing the amplification of primer–dimers and other nonspecific products.

Mastermixes without ROX or fluorescein

RT² SYBR Green qPCR Mastermix is highly suited for real-time PCR applications using SYBR Green-based detection on instrumentation not requiring a reference dye, including Bio-Rad models CFX96, CFX384; Bio-Rad/MJ Research Chromo4, DNA Engine Opticon, DNA Engine Opticon 2; Roche LightCycler 480 (96-well and 384-well); Eppendorf Mastercycler ep realplex without ROX filter set; and Cepheid SmartCycler.

Mastermixes with fluorescein

RT² SYBR Green Fluor qPCR Mastermix is highly suited for real-time PCR applications using SYBR Green-based detection on instrumentation that uses fluorescein as a reference dye, including the Bio-Rad models iCycler, iQ5, MyiQ, and MyiQ2.

Mastermixes with ROX

RT² SYBR Green ROX qPCR Mastermix is highly suited for real-time PCR applications using SYBR Green-based detection on instrumentation that uses ROX as a reference dye, including QIAGEN’s Rotor-Gene Q; Applied Biosystems models 5700, 7000, 7300, 7500 (Standard and Fast), 7700, 7900HT (Standard and Fast 96-well block, 384-well block), StepOnePlus, ViiA 7 (Standard and Fast 96-well block, 384-well block); Eppendorf Mastercycler ep realplex with or without ROX filter set; Stratagene models Mx3000P, Mx3005P, Mx4000; and Takara TP-800.

Applications

RT² SYBR Green qPCR Mastermixes are suitable for use in real-time PCR applications, especially RT² qPCR Primer Assays, RT² Profiler PCR Arrays, RT² lncRNA qPCR Assays, and RT² lncRNA PCR Arrays.

Supporting data and figures

High amplification efficiency over a wide dynamic range.

Resources

For pathway-focused gene expression analysis

For pathway-focused gene expression profiling using real-time RT-PCR

FAQ

HotStart PCR is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. Lack of sensitivity or specificity is most often caused by the amplification of nonspecific priming events, such as primer dimers, that usually occur at the lower temperatures when reactions are set up. Although thermostable DNA-dependent DNA polymerases have optimal activity at higher temperatures, they do also have some activity at lower temperatures when they may amplify these nonspecific priming events. HotStart enzymes are inactive at room temperature, and require heating at nucleic acid melting temperatures in order to be activated. In this way, nonspecific priming events are melted before the enzyme can amplify them. During PCR cycles, the temperature never drops low enough during annealing of gene-specific primers for nonspecific priming events to occur, resulting in amplification exclusively of the target of interest. When using a HotStart DNA polymerase, it is critical that the initial denaturation step in the experiment be of sufficient duration to fully activate the enzyme.

FAQ ID -2676

There are less than a dozen genes encoded by the mitochondrial genome (all other mitochondrial proteins are encoded by nuclear genes), and they are all transcribed as one transcript (just like any prokaryote), so distinguishing the expression of individual genes by real-time RT-PCR is not possible.

FAQ ID -2680

Be sure to carefully and completely seal the qPCR assay plate with fresh, optical, thin-wall, 8-cap strips or adhesive optical film before the plate is placed into the real-time cycler. In addition, refer to your instrument's user's manual to determine whether the real-time cycler manufacturer recommends use of a plate compression pad during the run.

FAQ ID -2679

There are several reasons for not seeing a PCR product.

1. The corresponding gene may not be expressed above the limit of detection of the qRT-PCR assay method.

2. There may have been experimental error, in which case, use a template known to contain the gene of interest as a positive control to troubleshoot the PCR reagents and experimental procedure.

3. The RNA may have been of poor quality, in which case, be sure to perform all of the recommended quality control checks on the RNA sample (see Sample Preparation FAQs, above).

4. There may not have been enough template, in which case, use more input total RNA, or use the template at a lower dilution factor (higher concentration), or use a larger volume of template.

5. Another possible explanation pertains to when one is trying to detect cellular expression from an exogenous vector that has been introduced into a cell. If the vector expresses only the open reading frame (ORF) of the gene of interest, and the qPCR primers being used amplify a target within the 5' or 3' UTR (untranslated region) of the gene, the transcript will not be detected.

FAQ ID -2685

You need:

A RT² SYBR Green Mastermix that matches the qPCR instrument in your laboratory;
RT² qPCR Primer Assays for your target genes;
A Housekeeping gene RT² qPCR Primer Assay.

We also recommend using our RT² First Strand Kit for reverse transcription.

FAQ ID -2707

The performance of our RT² qPCR Primer Assays have been tested, and are guaranteed with, our RT² SYBR Green Mastermixes only. Different master mixes have been optimized and are available for different qPCR instrumentation, because each instrument uses a different reference dye to normalize their optics. In order to guarantee that your RT² qPCR Primer Assays will work right the first time in your hands, we need to make sure that you receive the correct RT² SYBR Green Mastermix for your real-time instrument.

FAQ ID -2713

The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.

FAQ ID -2682

Our RT² qPCR Primer Assays may be used on any real-time instrument. qPCR solutions are available for the most popular qPCR instrumentation, including those from QIAGEN, ABI, BioRad, Stratagene.

Instrument-specific protocols are available for selected instruments, and can be accessed at the following link: http://www.sabiosciences.com/pcrarrayprotocolfiles.php

FAQ ID -2714

Assuming 100% amplification efficiency, each step increase in Ct value represents a doubling in the amount of qPCR template. Therefore, evaluating the difference in Ct values between the qPCR assay, and its matching NRT control, leads to the following predictions:

CtNRT - Ct+RT	Fraction of gene expression signal due to contaminating DNA	Percentage of gene expression signal due to contaminating DNA
1	(1/21) = 1/2	50%
2	(1/22) = 1/4	25%
3	(1/23) = 1/8	13%
4	(1/24) = 1/16	6%
5	(1/25) = 1/32	3%

FAQ ID -2688

You may be using too much template. Use less input total RNA for reverse transcription, or use template at a greater dilution factor (lower concentration). Do not pipet a volume of less than 1 μl.

FAQ ID -2684

Dissociation curves are carried out at the end of a PCR experiment by following a 3-step procedure.

First, all the components are denatured at 95°C, followed by complete annealing at a set temperature (based on the primer Tm values), followed by a gradual increase in temperature up to 95°C. Fluorescence intensity is monitored during this final temperature increase, resulting in the generation of a melting curve or dissociation curve.

By analyzing the first derivative of such a curve, you can readily assess the homogeneity of the PCR products, including the presence of primer–dimers, thereby determining the specificity of the PCR reaction. It is important to carry out such post-PCR analyses when using SYBR Green probe chemistry due to this reagent's lack of sequence specificity.

FAQ ID -2678

We can only guarantee the performance of RT² qPCR Primer Assays with our RT² SYBR Green Mastermixes. Our master mix components and primer design algorithm were optimized together to guarantee production of single bands of the predicted size. When we do test other sources of master mix with our Primer Assays, we frequently see primer dimers and other non-specific products that confound SYBR-Green based qPCR detection.

FAQ ID -2715

The sudden failure of a re-used RT² SYBR Green Mastermix is most likely due to repeated warming of the same 200-reaction scale product. We do not recommend repeatedly removing the same vial of PCR master mix from the -20 ºC freezer. The enzyme activity will decrease over time, under these conditions. Instead, upon receiving the master mix, divide into aliquots of a volume that you predict you will use for each day's experiment. Any unused portion of an aliquot should either be discarded or saved, noting that it has been used previously, for less critical uses or experiments. Make sure that you do not store the RT² SYBR Green Mastermixes frozen at -80 ºC, as this will kill master mix activity.

FAQ ID -2717

The performance of our RT² qPCR Primer Assays have been tested, and are guaranteed with, our RT² SYBR Green Mastermixes only. Our primer design algorithm accounts for our master mix buffer system parameters such as ionic strength and magnesium chloride concentration. We have not tested our Primer Assays with other sources of master mix and their buffer system parameters. We only guarantee that our primers will perform optimally with our master mixes. We do not guarantee optimal performance with other sources of master mix, without requiring further optimization studies by the end-user.

FAQ ID -2706

18S ribosomal RNA is a widely used control for qRT-PCR analyses because of its invariant expression across tissues, cells, and experimental treatments. However, due to its extremely high expression in most cell types, it can sometimes be challenging to use 18S rRNA as an endogenous normalizer for several gene expression assays in the same reaction.

FAQ ID -2675

The Rn value, or normalized reporter value, is the fluorescent signal from SYBR Green normalized to (divided by) the signal of the passive reference dye for a given reaction. The delta Rn value is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter reliably calculates the magnitude of the specific signal generated from a given set of PCR conditions. For more information, please refer to your cycler's user manual.

FAQ ID -2681

RT2 SYBR® Green qPCR Mastermixes

For use with RT2 Profiler, RT2 lncRNA, EpiTect ChIP, and EpiTect Methyl II PCR Arrays, and qPCR Assays

Products

RT² SYBR Green Fluor qPCR Mastermix (24)

RT² SYBR Green qPCR Mastermix (12)

RT² SYBR Green ROX qPCR Mastermix (24)

RT² SYBR Green qPCR Mastermix (2)

RT² SYBR Green ROX qPCR Mastermix (25 ml)

RT² SYBR Green Fluor qPCR Mastermix (12)

RT² SYBR Green qPCR Mastermix (24)

RT² SYBR Green ROX qPCR Mastermix (6)

RT² SYBR Green Fluor qPCR Mastermix (2)

RT² SYBR Green qPCR Mastermix (25 ml)

RT² SYBR Green ROX qPCR Mastermix (8)

RT² SYBR Green qPCR Mastermix (8)

RT² SYBR Green Fluor qPCR Mastermix (25 ml)

RT² SYBR Green Fluor qPCR Mastermix (6)

RT² SYBR Green ROX qPCR Mastermix (12)

RT² SYBR Green Fluor qPCR Mastermix (8)

RT² SYBR Green qPCR Mastermix (6)

RT² SYBR Green ROX qPCR Mastermix (2)

Features

Product Details

Performance

See figures

Principle

Mastermixes without ROX or fluorescein

Mastermixes with fluorescein

Mastermixes with ROX

Applications

Supporting data and figures

High amplification efficiency over a wide dynamic range.

Resources

FAQ

RT² SYBR^® Green qPCR Mastermixes

For use with RT² Profiler, RT² lncRNA, EpiTect ChIP, and EpiTect Methyl II PCR Arrays, and qPCR Assays