The SpyLine™ Poly A+ Capture Kit offers a simple, rapid, and cost-effective method for isolating poly A+ mRNA from cultured mammalian cells for direct RT-PCR. This kit features the SpyLine Poly A+ Plate, which is a 96-well PCR plate coated with oligo(dT) for selective capture of poly A+ mRNA from crude mammalian cell lysates. RT-PCR reagents can be added directly to this capture plate for subsequent RT-PCR cycling and analysis, or the mRNA can be eluted from the plate if desired. |
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| Lyse cells in culture plate |
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| Transfer lysate to capture plate |
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| Hybridize for 30-90 minutes |
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| Wash |
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| Perform RT-PCR directly in capture plate, or elute mRNA for other applications |
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Beneficial Features
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Sensitive
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Superior tool for knockdown measurement and gene expression analysis.
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Flexible
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Isolate mRNA from a wide variety of cell dilutions - even from a single cell.
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Consistent
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Extremely low well-to-well and plate-to-plate variability.
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Automatable
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96-well plate format suitable for use on high-throughput platforms.
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Convenient
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Capture and amplify mRNA in a single plate.
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Simple
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Rapid, easy-to-follow protocol.
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Ordering Information
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Product Code
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Product Name
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Available Sizes
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Technical Manual .pdf
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SpyLine Poly A+ Capture Kit |
1 x 96-well plate
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SpyLine Poly A+ Capture Kit |
4 x 96-well plate
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Superior sensitivity for siRNA knockdown
Figure 1. Target vs. dCT for SpyLine vs. Supplier Q. The figure above is a comparison of the change in Ct from the siRNA knockdown cells to the Non-Targeting siRNA cells. Plated HeLa cells were transfected with either siRNA transfection reagent mixture or non-targeting siRNA #1 reagent. After overnight incubation, the transfection media was removed, and the cells were lysed. One plate was purified by the SpyLine Poly A+ Capture Kit, while the other was purified using Supplier Q’s 96-well total RNA purification kit. Quantitative RT-PCR was run on the 2 sets of purified RNA using Applied Biosystems TaqMan® Gene Expression Assays.
Flexible enough for a variety of cell dilutions
Figure 2. Ct vs. cell number for decreasing amount of cells. A series of cell dilutions was prepared, beginning with 10,000 cells and decreasing by ten-fold increments down to 1 cell. SURF-4 mRNAs were then amplified by quantitative RT-PCR.
Figure 3. Quantitative RT-PCR of mRNA isolated from a series of decreasing cell dilutions. Cell dilutions were prepared in replicate, in decreasing 10-fold increments from 10000 cells to 1 cell. SURF-4 mRNAs were then amplified by quantitative RT-PCR. As depicted above, message can be clearly detected all of the way down to a single-cell input.
Consistent results - extremely low well to well and plate to plate variability
Figure 4. Well to well variability and plate to plate variability of the SpyLine Poly A+ Capture Kit. mRNA was isolated and captured utilizing the SpyLine standard protocol. Quantitative RT-PCR was set-up in the SpyLine Poly A+ Capture plate and was run targeting SURF-4 mRNA. Comparison of the two plates shows very little variability on each plate (standard deviation ~0.5 Cts), and very little variability from plate to plate (standard deviation ~0.5 Cts).
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