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ScreenTape® Enables Fast Quality Control of miRNA qPCR Reactions
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AnaSpec

The ScreenTape® System, developed by Lab901, employs a unique microscale polymer tape that contains all the reagents required for DNA electrophoresis. It brings major advantages of convenience, ease of use and safety to molecular biologists as absolutely no handling of gels and buffers is required. The compact, fully automated ScreenTape® instrument replaces the need for hand-cast gels, buffers, gel tanks and gel-doc systems. In just under 10 minutes the system loads the samples, runs the gel, images and presents the fully analysed results (including gel picture, band sizes and an indication of quantitative data).This ensures accuracy, reproducibility and traceability. The system is fully bar-coded to meet GLP guidelines and data is archived for easy retrieval and comparison.

Quantitative PCR (qPCR) is routinely used to assess miRNA expression levels. Quality control of the reaction by gel electrophoresis confirms the presence of a single product of the correct size. This control can be achieved using ScreenTape, within 10 minutes, providing automated, hands-free analysis without reagent or gel preparation.

Introduction

Following qPCR analysis, it is important to check that the reaction only contains amplified products that are specific to the chosen primers. This is routinely achieved by melting curve analysis on the qPCR instrument. However, this method is not always precise as it often distinguishes poorly contaminating bands with small size or sequence differences. Many scientists therefore prefer to use traditional gel electrophoresis. ScreenTape provides a novel and more efficient method of analysing and precisely sizing qPCR products.

Materials and Methods used in qPCR

Purified RNA from MCMV infected macrophages and 3T3 cells were poly-A tailed and reverse transcribed using the NCode™ miRNA First strand cDNA synthesis kit (Invitrogen), following the manufacturer’s instructions. SYBR green PCR was performed using qPCR supermix-UDG™ (Invitrogen) and conditions were optimized to generate only one product of around 65-70bp for each primer in infected cells, whereas a product of 44bp was observed for uninfected cells, showing the presence or absence of a specific regulating viral miRNA.

ScreenTape DNA Analysis Procedure

Samples were mixed 1:1 with loading buffer and placed in the TapeStation™ with pre-packaged ScreenTape. After clicking “START” on the software driven menu, full analysis and archiving of the results was achieved with no user intervention within 10 minutes.

Results on ScreenTape

Analysed results shown in Figure 1 immediately provide band sizes for each of the qPCR reactions. Results agree with the gel and dissociation curve analysis seen in Figure 2. Sample 1 shows a product of 44bp diagnostic of an uninfected sample, whereas samples 2 in macrophages and 3 in 3T3 cells have a product of 65bp, indicating that they are from MCMV infected cells.

Figure 1: Results from qPCR products run on ScreenTape showing amplified MCMV miRNA from infected 3T3 cells in lane 2 and from infected macrophages in lane 3. Lane 1 shows a result from un-infected 3T3 cells. An electropherogram of sample 2 (highlighted green) is presented on the right hand side.

Figure 2: Results from miRNA qPCR reactions run on an agarose gel showing an uninfected sample in lane 2 and MCMV infected 3T3 cells and macrophages in lanes 3 and 4 respectively. Dissociation curves of these PCR products can be seen in the associated graph.

Benefits of using ScreenTape for qPCR QC.

Like the dissociation curve analysis, ScreenTape provides the convenience of an automated solution to qPCR QC with the precision and certainty of slab gel analysis. Band sizes are accurately and automatically determined, with minor contaminants being easily identified. ScreenTape offers significant time saving for gel analysis of qPCR reactions, by providing a fully automated, walk-away solution. ScreenTape electrophoresis is convenient and integrates seamlessly with qPCR analysis. As no reagent preparation is needed and Ethidium Bromide and UV light are eliminated; user-safety is enhanced. The TapeStation instrument is no bigger than a desk-top printer which saves space and keeps the lab bench free of spilt buffers.

These results have recently been published: Buck AH, Santoyo-Lopez J, Robertson KA, Kumar DS, Reczko M, Ghazal P. (2007). Discrete clusters of virus-encoded micrornas are associated with complementary strands of the genome and the 7.2-kilobase stable intron in murine cytomegalovirus. J Virol. Dec;81(24):13761-70.



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