MessageBOOSTER™ cDNA Synthesis kit for qPCR

The MessageBOOSTER™ cDNA Synthesis Kit for qPCR greatly improves qRT-PCR sensitivity, accuracy, and reproducibility of even low-abundance transcripts from as little as 1 cell. A MessageBOOSTER Kit reaction amplifies the Poly(A)RNA in a total RNA preparation from 1 to 50 cells (approximately 10 pg to 500 pg of total RNA) and then converts the amplified RNA to single-stranded sense cDNA that is ready for qPCR. Poly(A) RNA amplification and subsequent cDNA synthesis is achieved using a simplified and improved "Eberwine" linear RNA amplification process (Figure 1) that maintains the gene expression profile of the sample.

The number of qPCR that can be obtained using cDNA produced by a MessageBOOSTER reaction is dependent on two factors:

The amount of total RNA added to the MessageBOOSTER reaction.
The abundance of the transcript(s) of interest.

Table 1 summarizes our estimates of the number of real-time qPCR reactions that can be obtained using cDNA produced from MessageBOOSTER reactions containing input total RNA from ~ 1, 10, and 50 Normal Rat Kidney (NRK) cells.

Figure 1. A MessageBOOSTER™ reaction utilizes a linear RNA amplification process to amplify the poly(A) RNA (mRNA) from total RNA of 1 to 50 cells (10 pg to 500 pg total RNA) and then coverts the aRNA produced to cDNA that is ready for qPCR.

Applications

Quantitative real-time RT-PCR (qRT-PCR) using total RNA from 1 to 50 cells (approximately 10 pg to 500 pg of total RNA)

Benefits

Produces cDNA for sensitive and reproducible qPCR of even low-abundance transcripts (mRNA) from total RNA of 1 to 50 cells (Figure 2).
Linear RNA amplification process preserves the gene expression profile.
Each reaction produces enough cDNA for up to 10 qPCR reactions for low-abundance transcripts from total RNA of 1 cell (approximately 10 pg total RNA).
Enables multiplex qRT-PCR using total RNA from 1 cell (Figure 3).
MMLV Reverse Transcriptase is provided in the kit and reactions are readily adapted for use with SuperScript™ III Reverse Transcriptase (provided by the user) if desired.

Figure 2. cDNA produced from 10 pg of total NRK RNA using the MessageBOOSTER™ cDNA Synthesis Kit for qPCR significantly improves the sensitivity (lowers the CT) of detecting low-, medium- and high-abundance transcripts compared to cDNA produced from 10 pg of unamplified RNA.

Low-abundance transcript PBGD

Low- to medium-abundance transcript PBX2

Medium- to high-abundance transcript B2M

Table 1. The Number of qPCR reactions that can be performed using cDNA produced by a MessageBOOSTER™ reaction is dependent on the amount of input total RNA and the abundance of the transcript(s) of interest.

Amount of Total RNA in a MessageBOOSTER™ Reaction.	Number of Real-Time qPCR Reactions That Can Be Performed.
Amount of Total RNA in a MessageBOOSTER™ Reaction.	Low- to Medium- Abundance Transcripts*	Medium- to High- Abundance Transcripts*
10 pg (~1 cell)	>10	>100
100 pg (~10 cells)	>100	>1,000
500 pg (~50 cells)	>500-1,000	>5,000-10,000

*Low-Abundance Transcripts = 1-1,000 copies per cell

Medium-Abundance Transcripts = 1,000-10,000 copies per cell

High-Abundance Transcripts = 10,000-100,000 copies per cell

Figure 4. A MessageBOOSTER™ cDNA Synthesis Kit for qPCR reaction preserves the relative transcript abundance of the sample.

4A) the relative transcript abundance of 20 mRNA targets in Human Reference RNA and the levels in adult skeletal muscle RNA before and after a MessageBOOSTER reaction. 4B) Scatter plot of the data of Figure 3A. R2=0.997

Figure 4A.

Figure 4B.

Message BOOSTER is a trademark of EPICENTRE Biotechnologies. SuperScript is a trademark of Invitrogen Corp., Carlsbad, CA.

商品名	Catalog No	Size
MessageBOOSTER™ cDNA Synthesis Kit for qPCR	MB060110	10 Reactions
MessageBOOSTER™ cDNA Synthesis Kit for qPCR	MB060124	24 Reactions

製品のご購入・ご相談はTEL:03-3545-5720、またはお問い合わせまで。