Short video answers to questions about the direct lysis approach to DNA/RNA analysis, by Richard Fekete, Ph.D Senior Manager R&D

  Can I use 48-, 24-, or 6-well plates for direct cell lysis prior to qPCR?     Can I use cells that are frozen or stabilized with RNAlater® solution?
             
  Is it possible to freeze the lysates at -80oC, and for how long?     Can I use the direct lysis solution for uncultured primary cells or my other cell lines?
             
  Can I automate the rapid lysate method?     Can I  also use heparin or EDTA-treated blood?
             
  Does the product simultaneously isolate DNA and RNA?     Can I use the rapid lysate method for tissue samples?
             
  How do I know if there is RT inhibition?     Would the direct lysis approach be a good alternative for very small samples?
             
  How do I assess RNA quality with a lysate based method?     Is the method applicable for extraction of viral RNA?
             
  Can the rapid lysate method be used for bacterial RNA extraction?        

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
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?

  1. Ensure all media is removed from the wells.
  2. Wash with an equal volume of room temperature 1X PBS after the media removed.
  3. 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).
  4. Warm lysis solution to room temperature before adding to cells
  5. Allow lysis reaction to proceed for 8 minutes.
  6. Perform lysis reaction at 25°C for up to 8 minutes.