HIGH SENSITIVITY QRT-PCR

HIGH SENSITIVITY qRT-PCR

MessageSensor™ Reverse Transcription Kit For One Step qRT-PCR

• Linear quantitation of mRNA targets over a 106-fold range

• Quantitate transcripts from as little as 500 fg total RNA – 1/20th of a single cell!

• Fast, flexible RT step complete in 15 min

• Use reaction temperatures up to 50°C

• Compatible with TaqMan^®, SYBR^® Green, and gel-based detection methods

RNase H^– RT Reduces Detection Sensitivity in RT-PCR
RNase H minus (RNase H^–) reverse transcriptases (RTs) are a popular choice among researchers for applications that require a cDNA synthesis step. RNase H^– RTs are available from several manufacturers, with SuperScript II (Invitrogen) being the most widely used. RNase H^– RTs can generate longer cDNA products with higher yields than wild type (RNase H⁺) RTs, which are important criteria when generating cDNA libraries or performing DNA microarray analysis. However, these benefits are unnecessary for qRT-PCR where amplicons average 100 bp. Additionally, RNase H^– RT can limit the sensitivity of qRT-PCR detection (Figures 1 and 2). If the RNA template is not degraded after first strand cDNA synthesis, it can bind to the newly-synthesized cDNA and restrict its accessibility to primers during subsequent PCR amplification. RNase H mediated destruction of the template can prevent this problem and improve the sensitivity of RT-PCR analysis.

Recently, Polumuri and colleagues tested the effects of RNase H treatment on RT-PCR detection sensitivity using SuperScript II MMLV RNase H^– RT to amplify three genes (NCX1, NCX2, and NCX3) (BioTechniques 32: 1224–1225 (2002)). Of these three targets, one (NCX2) was detected much more readily when an RNase H step was included after the reverse transcription. Experiments at Ambion have also shown that better overall qRT-PCR sensitivity can be obtained by either using an RNase H⁺ RT for reverse transcription (Figure 1), or by including an RNase H treatment step after reverse transcription with an RNase H^– RT. These results strengthen the general finding that RNase H treatment is desirable for the most sensitive detection of RT-PCR products.

Ambion's MessageSensor RT Kit includes an MMLV RT that maintains full RNase H activity. Figure 2 shows that the MessageSensor RT clearly outperforms a similar kit that uses RNase H^– RT. For all amplicons tested, use of MessageSensor resulted in C_t values that were 2–6 cycles lower compared to the competitor's kit. This represents an up to 64-fold increase in sensitivity of detection. For a comprehensive listing of real-time RT-PCR results comparing MessageSensor to SuperScript™ II, click here.

Figure 1. MessageSensor™ RNase H⁺ RT vs. an RNase H^– RT Containing Kit. Primers to the Na⁺/Ca²⁺ exchanger gene NCX2 were used to perform RT-PCR on 100 ng of Rat Brain Total RNA (Ambion). The amplicon is 452 bases. After 24 cycles, samples were loaded on a 1.5% agarose gel and stained with EtBr. Duplicate reactions were performed for each RT-PCR kit. RT minus controls were negative for both kits.

Figure 2. One Step RT-PCR With MessageSensor™ System and a Popular MMLV RT RNase H^– System. C_t values associated with cdc-2 and ß-actin targets were detected from 10 pg HeLa-S3 total RNA, whereas c-jun was detected from 100 ng HeLa-S3 total RNA. Note: A lower C_t value indicates a more sensitive detection system. 40 cycles represents no product detected (ND).

MessageSensor: RNA to PCR Results in 2 hrs
MessageSensor provides unsurpassed results in the least amount of time. The kit contains a unique formulation of MMLV RT developed for a one tube real-time RT-PCR approach to create a highly optimized protocol and reagent system. The result is a kit that delivers rapid results without compromising lower-end sensitivity. Simply prepare a master mix that combines your RNA sample, primers, and dye-based reporter. Add Taq DNA polymerase, with the MessageSensor RNase H⁺ reverse transcriptase, buffer and nucleotides, and you are ready to go. The reverse transcription step takes only 15 minutes -- you can go from RNA sample to results in less than 2 hours. And although the protocol is short, MessageSensor won't compromise your limit of detection: human GAPDH mRNA can be readily detected from as little as 500 fg of total RNA-- about 1/20th of the total RNA in a single cell.

Linear Detection Over a Million-fold Range of Input RNA
Effective qRT-PCR requires the reliable detection of targets over a broad range of input RNA amounts. Figure 3 demonstrates that the amplification reaction produces linear results (r²=0.998; triplicate samples) across a million-fold range of inputs – 500 fg to 0.5 µg of total RNA – when human GAPDH mRNA is amplified using MessageSensor. Linear standard curves can also be obtained using the included SYBR Green Buffer that enables DNA detection with SYBR Green dye (not included). In contrast to other kits that require different enzymes for low and high amounts of input RNA, MessageSensor provides a one-size-fits-all solution for your most demanding qRT-PCR applications. Real-time or end point detection and quantitation of mRNA targets can be performed on poly(A) RNA, total RNA, or synthetic transcripts.

Figure 3. Linear Quantitation Over 106-fold Range of Input RNA with MessageSensor™. One step RT-PCR was performed with a triplicate standard curve of 500 fg to 0.5 µg HeLa-S3 total RNA. The target was Human GAPDH, detected using a TaqMan^® probe. (A) Standard curve, r²=0.998; slope=-3.4.(B) Amplification plot, showing triplicate data. All RT minus controls and RNA minus controls were negative.

A Complete RT Kit for RT-PCR Applications
Unlike many qRT-PCR kits, MessageSensor includes a total RNA control, a control human GAPDH primer set, RNase inhibitor, and nucleotides, as well as a buffer additive that enables DNA detection with SYBR Green dye. The MessageSensor Kit contains reagents for 50 reactions. By purchasing the SuperTaq™ Real-Time and MessageSensor Kits together, you can get all the components you need for one-step qRT-PCR (except for gene-specific primers and fluorogenic probe or dye) in a single convenient order.

Figure 4. MessageSensor™ MMLV RT Retains Full Activity at 50°C. Reverse Transcription reactions were performed at 42°C or 50°C for 15 min, followed by real-time PCR using TaqMan^® probes. C_t values for
cdc-2, ß-actin, and GAPDH were detected from 10 pg HeLa-S3 total RNA.

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Ordering Information
Cat# Product Name Size 1745 MessageSensor™ RT Kit 50 rxn