RNA extraction or direct lysis –the most frequently asked questions
You don’t have to isolate RNA to analyze gene expression. With the Cells-to-CT™ approach, you simply lyse the cells - with minimal processing - into a solution that is compatible with downstream RT-PCR, without the need for RNA purification.
In this video series, Senior R&D Manager Richard Fekete, Ph.D., presents short answers to typical questions related to the direct lysis approach to RNA analysis and RT-qPCR.
Ambion® Cells-to-CT™ frequently asked questions
1). 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 |
HEK-293 | adherent | H. sapiens | Kidney |
COS-7 | adherent | C. aethiops (monkey) | Kidney |
CHO-K1 | adherent | C. griseus (hamster) | Ovary |
NCI-H460 | adherent | H. sapiens | Lung |
DU-145 | adherent | H. sapiens | Prostate |
K562 | suspension | H. sapiens | Bone Marrow |
U-2 OS | adherent | H. sapiens | Bone |
Huh-7 | adherent | H. sapiens | Liver |
Neuro 2A | adherent | M. musculus | Brain |
BJ | adherent | H. Sapiens | Foreskin |
2). 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.
3). Why do you say this is a “green’ alternative to standard purification?
Less waste, less hazardous - 10 Sample Comparison
RNeasy™ 35 minutes 140 g plastic waste 18 mL hazardous waste |
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Cells-to-CT™ 7 minutes 6.6 g plastic waste 0 mL hazardous waste |
4). How do I remove gDNA from my 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.