1. How do Cells-to-CT kits work?
Cells-to-CT kitsprovide a complete workflow for real-time qRT-PCR analysis directly from cultured cells without RNA purification. A 1-step reaction is when RT and qPCR are performed in a single well. In the qPCR step, TaqMan Assays use custom primer and probe combinations designed for each target. These assays are more sensitive and specific than SYBR Green assays, which use generic qPCR primers. The kits uses reverse transcription (RT) reagents for cDNA synthesis and is compatible with dye-based (SYBR Green dye) or probe-based (TaqMan probes) master mixes for real-time PCR analysis.
The 1-step and 2-step kits differ in that the 1-step kits combine reverse transcription and real-time PCR into a single step, reducing overall protocol time.
2. Which cell lines have been tested?
This is a short list of the cell lines compatible with the Cells-to-CT system.
|Cell Line||Growth Type||Source Species||Source Tissue|
|HeLa||adherent||H. sapiens||Cervical Adenocarcinoma|
|HepG2||adherent||H. sapiens||Liver Carcinoma|
|Primary Hepatocytes||adherent||H. sapiens||Liver|
|SK-N-AS||adherent||H. sapiens||Brain Neuroblast|
|SK-N-SH||adherent||H. sapiens||Brain Fibroblast|
|U-87 MG||adherent||H. sapiens||Brain Glioblastoma|
|ME-180||adherent||H. sapiens||Cervical Epidermoid Carcinoma|
|A549||adherent||H. sapiens||Lung Carcinoma|
|Jurkat||suspension||H. sapiens||Acute T-Cell Leukemia|
|PC-12||adherent||R. norvegicus (rat)||Adrenal Pheochromocytoma|
|PT-K75||adherent||S. scrofa (pig)||Nasal Turbinate Mucosa|
|NIH/3T3||adherent||M. musculus (mouse)||Embryonic Fibroblast|
|Raji||suspension||H. sapiens||B Lymphocyte|
|COS-7||adherent||C. aethiops (monkey)||Kidney|
|CHO-K1||adherent||C. griseus (hamster)||Ovary|
|K562||suspension||H. sapiens||Bone Marrow|
|U-2 OS||adherent||H. sapiens||Bone|
|Neuro 2A||adherent||M. musculus||Brain|
3. Will it work with my special cell line?
There’s no reason why the Cells-to-CT system shouldn’t work with any cell line. (Please refer to the table above for cell lines tested and confirmed to be compatible). However, due to differences in cell size and composition, the maximum number of cells per lysis reaction may be slightly different for different cell lines. Testing for inhibition and minimal sample input by using he TaqMan Cells-to-CT Control Kit.
4. Why do you say this is a "green" alternative to standard purification?
Less waste, less hazardous - 10 Sample Comparison
140 g plastic waste
18 mL hazardous waste
6.6 g plastic waste
0 mL hazardous waste
5. How to isolate genomic DNA from a Cells-to-CT reaction?
- Ensure all media is removed from the wells.
- Wash with an equal volume of room temperature 1X PBS after the media removed.
- Ensure reaction happens at room temperature (lysis reaction may not reach room temperature if plate is on ice, quickly moved to bench, or cold lysis solution is added).
- Warm lysis solution to room temperature before adding to cells
- Allow lysis reaction to proceed for 8 minutes.
- Perform lysis reaction at 25°C for up to 8 minutes.
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