Ten Tips for Getting the Most Out of the TaqMan® Gene Expression Cells-to-CT™ Kit
| 1 | Performing DNA Analysis on Lysates | The TaqMan® Gene Expression Cells-to-CT™ Kit can be used to purify DNA for analysis. Just omit the DNase from the Lysis Solution before adding it to your samples. Then use the Stop Solution as you would normally, according to the kit instructions. Typically, the maximum number of cells you can lyse is 10,000, due to the viscosity of the lysates. Perform a test lysis on an aliquot of the cells you’re planning to use, to check that the viscosity is acceptable. Up to 10% of the volume of the PCR reaction can be lysate, and the lysates produced with the TaqMan® Gene Expression Cells-to-CT™ Kits are compatible with Universal, Gene Expression, and Genotyping Master Mixes. |
| 2 | Purified Lysates | If you need purified RNA for an application other than TaqMan® gene expression analysis, you can easily process the lysates using RNAqueous® kits. Just add the Lysis/Binding Solution from the RNAqueous® kit and follow that kit’s protocol to complete the purification. |
| 3 | Lysing Cells on a Larger Scale | For cells growing in tissue culture plates larger than 96-well dishes, you’ll need to use more Lysis Solution to ensure complete lysis. Be sure to add enough Lysis Solution so that the entire well surface is covered and there are no more than 100,000 cells per 50 µL of Lysis Solution. Scale up the amount of DNase and Stop Solution proportionally to the amount of Lysis Solution you use. |
| 4 | gDNA Removal | The Cells-to-CT™ kits were optimized to remove gDNA from cells, but some cell lines have more gDNA than others. If you are working with a cell line that is particularly prone to gDNA contamination, the first thing to note is that adding more DNase is not beneficial. What can help is making sure that all of the medium is removed from the cells. After the medium is removed, wash with an equal volume of room-temperature 1X PBS. Make sure the Lysis Solution is at room temperature before adding it to your cells. Also, ensure that the lysis reaction happens at room temperature (plates that have just been removed from ice have not had sufficient time to reach room temperature), or the lysis reaction can be raised to 25°C if needed. You can also extend the duration of the lysis to 8 minutes. |
| 5 | Adding TaqMan® Arrays to Your Investigation | To get a broader view of gene expression using qRT-PCR, TaqMan® Arrays are a great option. Follow the same procedure for cell lysis and reverse transcription, and then just substitute the cDNA made from cell lysates for the cDNA called for in the TaqMan® Array protocol. Up to 45% of the sample loaded into each port of the TaqMan® array can be cDNA from a lysate, allowing for greater sensitivity in signal. |
| 6 | Preventing Foaming in Lysates | When mixing the lysates with a pipettor, do not depress the piston to the “blow out” position. This will prevent air from being added to the lysates, which can lead to foaming and difficulty when pipetting later. |
| 7 | DNase Stability in Lysis Solutions | After mixing the DNase into the Lysis Solution, you do not need to keep it on ice. The mixture is stable at room temperature for up to an hour. |
| 8 | Longer-Term Storage of Stop Solution | For medium- to high-throughput applications, the Stop Solution can be dispensed in aliquots into an 8-tube strip or 96-well plate for easier distribution into 96-well plates. Any leftover Stop Solution can be frozen in the aliquots and used again for the next experiment. The Stop Solution can withstand up to 5 freeze/thaw cycles. |
| 9 | Mixing May Be Optional | When using liquid handlers, the mixing steps (after adding the Lysis and Stop Solutions) can be difficult to optimize. Using 4,000 plated HeLa cells, we tested the effect on Ct of mixing 5 times (recommended procedure), not mixing, and shaking the plate for 30 seconds after the Lysis Solution was added. Very similar Ct values were obtained for all conditions. The reason for this is that the liquid handler puts the Stop Solution into the center of the well, so that all of the Stop Solution is added. We recommend testing the no-mix procedure in your experiment to see if you get the same results as when you mix. |
| 10 | Ensuring Maximum Reproducibility | To help ensure reproducible data from your replicates, completely remove the PBS wash from your cells, and add the Stop Solution directly into the cell lysate. If PBS is left behind, the amount can vary from well to well and, if more than 5 µL is left, it can reduce the effectiveness of the Lysis Solution. The Stop Solution needs to be added into the lysate to fully inactivate the Lysis Solution. If the Stop Solution is added to the side of the tube/well, it may not get fully incorporated into the lysate sample. |
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