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